Halogenated rhodamine derivatives and applications thereof

ABSTRACT

Methods for treating an infection in a tissue sample are described. Tissue samples that are harvested from a patient may be infected with either bacteria or a virus. A rhodamine compound is mixed with the infected tissue sample to form a mixture. The mixture is then exposed to radiant energy to inhibit or kill the bacteria. The exposed mixture is then transplanted into the patient. The rhodamine compounds may be 2′-(6-dimethylamino-3-dimethylimino-3H-xanthen-9-yl) 4′, 5′-dichloro-benzoic acid methyl ester hydrochloride.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 13/157,105, filedJun. 9, 2011, which is a continuation of U.S. application Ser. No.12/786,280, filed May 24, 2010, now abandoned, which is a continuationof U.S. application Ser. No. 12/403,819, filed Mar. 13, 2009, nowabandoned, which is a division of U.S. application Ser. No. 10/297,088,filed May 30, 2003, now issued as U.S. Pat. No. 7,560,574, which in turnis a national stage application of international application no.PCT/CA02/00438, filed Mar. 27, 2002, which in turn claims priority toCanadian Application No. 2,342,675 and U.S. application Ser. No.09/822,223, both filed Apr. 2, 2001. All of the above applications areexpressly incorporated herein by reference in their entirety for allpurposes.

FIELD OF THE INVENTION

The invention relates to new rhodamine derivatives that are useful fortheir pharmaceutical and non-pharmaceutical properties.

The rhodamine derivatives of the invention exhibit powerful bactericidaland antiviral activities.

They are also useful, alone or in association with a pharmaceuticallyacceptable carrier, in the treatment and/or in the prevention ofimmunologic disorders.

Moreover, those derivatives are useful as intermediates I the synthesisof further new rhodamine derivatives and also in the new synthesis ofalready known rhodamine derivatives.

Finally, the present invention also relates to new processes for thepreparation of rhodamine derivatives.

BACKGROUND OF THE INVENTION

Photodynamic therapy has been used as a method for the eradication ofneoplastic cells from autologous grafts for cancer treatments. Thismethod relies on the use of photosensitizing dyes, which when activatedwith light of a particular wavelength, produce toxic O₂ radicals,ultimately leading to cell death. Photochemical treatments have alsobeen used for pathogen inactivation, such as in “decontamination” ofblood and blood-derived products. The danger of pathogen transmissionthrough transfusion of whole blood, platelets concentrates, plasmaand/or red blood cells still represent major concerns in medicine.Although there has been impressive progress in the prevention andmaintenance of blood safety regarding the presence of microorganisms,blood components continue to carry risk of pathogen transfusion.Moreover, the presence of viruses in blood components is also of greatconcerns, mainly for the presence of Hepatitis C and humanimmunodeficiency virus (HIV), even though the risk of contamination isreduced to negligible levels. The presence of other viruses is alsorequired and includes the human T-cell lymphotrophic virus type 1(HTLV-1), Hepatitis B (HBV) and cytomegalovirus. Photodynamic compoundssuch as pseuralens, porphyrines, riboflavines and dimethyl of methylenebleue have been used in the treatment of pathogen in blood product.These compounds necessitate radiation by a ultra violet A lamp (UVA) toget activated, thus leading to possible mutagenic effect in theremaining cells present in the treated samples. (Corash, L.,Inactivation of infectious pathogens in labile blood components: meetingthe challenge, Transfus Clin Biol, 2001, 8, 138-145 Lin, L., Londe, H.,Janda, M. J., Hanson, C. V. and Corash, L., Photochemical inactivationof pathogenic bacteria in human platelet concentrates, Blood, 1994, 83,9, 2698-2706; Lin, L, Londe, H., Hanson, C. V., Wiesehahn, G., Isaacs,S., Cimino, G. and Corash, L., Photochemical inactivation ofcell-associated human immunodeficiency virus in platelet concentrates,Blood, 1993, 82, 1, 292-297; Lin, L., Eiesehahn, G. P., Morel, P. A. andCorash, L., Use of 8-methoxypsoralen and long-wavelength ultravioletradiation for decontamination of platelet concentrates, Blood, 1989, 74,1, 517-525; Lin, L., Cook, D. N., Wiesehahn, G. P., Alfonso, R.,Behrman, B., Cimino, G. D., Corten, L., Damonte, P. B., Dikeman, R.,Dupuis, K., Fang. Y. M., Hanson, C. V., Heasrt, J. E., Lin, C. Y.,Londe, H., Metchette, K., Nerio, A. T., Pu, J. T., Reames, A. A.,Rheinschmidt, M., Tessman, J., Isaacs, S. T., Wollowitz, S. and Corash,L., Photochemical inactivation of viruses and bacteria in plateletconcentrates by use of a novel psoralen and long-wavelength ultravioletlight, Transfusion, 1997, 37, 423-435). Because of the UVA exposure toblood components, these techniques are not entirely satisfactory. Therewas therefore a need for new light sensitive derivatives that do notnecessitate UVA exposure of blood components and that may also be asafer ad more acceptable replacement to UVA treated blood components.

Immunologic disorders are uncontrolled cell proliferations that resultfrom the production of immune cells recognizing normal cells and tissuesas foreign. After a variable latency period during which they areclinically silent, cells with immunoreactivity towards normal cellsinduce damages in these normal cells and tissues. Such immunologicdisorders are usually divided in alloimmune conditions and autoimmuneconditions. Alloimmune disorders occur primarily in the context ofallogeneic transplantation (bone marrow and other organs: kidney, heart,liver, lung, etc.). In the setting of bone marrow transplantation, donorimmune cells present in the hematopoietic stem cell graft react towardshost normal tissues, causing graft-versus-host disease (GVHD). The GVHDinduces damage primarily to the liver, skin, colon, lung, eyes andmouth. Autoimmune disorders are comprised of a number of arthriticconditions, such as rhumatoid arthritis, scleroderma and lupuserythematosus; endocrine conditions, such as diabetes mellitus;neurologic conditions, such as multiple sclerosis and myasthenia gravis;hematological disorders, such as autoimmune hemolytic anemia, etc. Theimmune reaction, in both alloimmune and autoimmune disorders, progressesto generate organ dysfunction and damage.

Despite important advances in treatment, immunologic complicationsremain the primary cause of failure of allogeneic transplantations,whether in hematopoietic stem cell transplantation (GVHD) or in solidorgan transplantation (graft rejection). In addition, autoimmunedisorders represent a major cause of both morbidity and mortality.Prevention and treatment of these immune disorders has relied mainly onthe use of immunosuppressive agents, monoclonal antibody-basedtherapies, radiation therapy, and more recently molecular inhibitors.Significant improvement in outcome has occurred with the continueddevelopment of combined modalities, but for a small number of disordersand patients. However, for the most frequent types of transplantations(bone marrow, kidney, liver, heart and lung), and for most immunedisorders (rheumatoid arthritis, connective tissue diseases, multiplesclerosis, etc.) resolution of the immunologic dysfunction and cure hasnot been achieved. Therefore, the development of new approaches for theprevention and treatment of patients with immunologic disorders iscritically needed particularly for those patients who are at high riskor whose disease has progressed and are refractory to standardimmunosuppressive therapy. Allogeneic stem cell transplantation(AlloSCT) has been employed for the treatment of a number of malignantand non-malignant conditions. Allogeneic stem cell transplantation isbased on the administration of high-dose chemotherapy with or withouttotal body irradiation to eliminate malignant cells, and hosthematopoietic cells. Normal hematopoietic donor stem cells are theninfused into the patient in order to replace the host hematopoieticsystem. AlloSCT has been shown to induce increased response rates whencompared with standard therapeutic options. One important issue thatneeds to be stressed when using AlloSCT relates to the risk ofreinfusing immune cells that will subsequently recognize patient cellsas foreign and cause GVHD. A variety of techniques have been developedthat can deplete up to 10⁵ of T cells from the marrow or peripheralblood. These techniques, including immunologic and pharmacologicpurging, are not entirely satisfactory. One major consideration whenpurging stem cell grafts is to preserve the non-host reactive T cells sothat they can exert anti-infectious and anti-leukemia activity upongrafting. The potential of photodynamic therapy, in association withphotosensitizing molecules capable of destroying immunologicallyreactive cells while sparing normal host-non-reactive immune cells, topurge hematopoietic cell grafts in preparation for AlloSCT or autologousstem cell transplantation (AutoSct), and after AlloSCT in the context ofdonor lymphocyte infusions to eliminate recurring leukemia cells haslargely been unexplored. To achieve eradication of T cells, severalapproaches have been proposed including:

-   -   1) in vitro exposure of the graft to monoclonal antibodies and        immunotoxins against antigens present on the surface of T cells        (anti-CD3, anti-CD6, anti-CD8, etc.);    -   2) in vitro selection by soybean agglutinin and sheep red blood        cell rosetting;    -   3) positive selection of CD34+ stem cells; and    -   4) in vivo therapy with combinations of anti-thymocyte globulin,        or monoclonal antibodies.    -   5) In vitro exposure of recipient-reactive donor T cells by        monoclonal antibodies or immunotoxins targeting the interleukin        2 receptor or OX-40 antigen (Cavazzana-Calvo M. et al. (1990)        Transplantation, 50:1-7; Tittle T. V. et al (1997) Blood        89:4652-58; Harris D. T. et al. (1999) Bone Marrow        Transplantation 23:137-44).

However, most of these methods are not specifically directed at thealloreactive T cell subset and associated with numerous problems,including disease recurrence, graft rejection, second malignancies andsevere infections. In addition, the clinical relevance of several ofthese methods remains to be established.

There are many reports on the use of photodynamic therapy in thetreatment of malignancies (Daniell M. D., Hill J. S. (1991) Aust. N. Z JSurg., 61: 340-348). The method has been applied for cancers of variousorigins and more recently for the eradication of viruses and pathogens(Raab O. (1990) Infusoria Z. Biol., 39:524).

The initial experiments on the use of photodynamic therapy for cancertreatment using various naturally occurring or synthetically producedphotoactivable substances were published early this century (JesionekA., Tappeiner V. H. (1903) Muench Med Wochneshr, 47: 2042; Hausman W.(1911) Biochem. Z., 30: 276). In the 40's and 60's, a variety of tumortypes were subjected to photodynamic therapy both in vitro and in vivo(Kessel, David (1990) Photodynamic Therapy of neoplastic disease, Vol.I, II, CRC Press. David Kessel, Ed. ISBN 0-8493-5816-7 (v. 1), ISBN0-8493-5817-5 (v. 2)). Dougherty et al. and others, in the 70's and80's, systematically explored the potential of oncologic application ofphotodynamic therapy (Dougherty T. J. (1974) J Natl Cancer Inst., 51:1333-1336; Dougherty T. J. et al. (1975) J. Natl Cancer Inst., 55:115-121; Dougherty T. J. et al. (1978) Cancer Res., 38: 2628-2635;Dougherty T. J. (1984) Urol. Suppl., 23: 61; Dougherty T. J. (1987)Photochem. Photobiol., 45: 874-889).

Treatment of Immunoreactive Cells with Photodynamic Therapy

There is currently a lack of agents which allow selective destruction ofimmunoreactive cells while leaving intact the normal but suppressedresidual cellular population. Preferential uptake of photosensitive dyeand cytotoxicity of photodynamic therapy against leukemia (Jamieson C.H. et al. (1990) Leuk. Res., 14: 209-219) and lymphoid cells (Greinix H.T., et al. Blood (1998) 92:3098-3104; are reviewed in Zic J. A. et al.Therapeutic Apheresis (1999) 3:50-62) have been previously demonstrated.

It would be highly desirable to be provided with photosensitizers whichpossess at least one of the following characteristics:

-   -   i) preferential localization and uptake by the immunoreactive        cells;    -   ii) upon application of appropriate light intensities, killing        those cells which have accumulated and retained the        photosensiting agents;    -   iii) sparing of the normal hemopoietic stem cell compartment        from the destructive effects of activated photosensitizers; and    -   iv) potential utilization of photosensitizers for hematopoietic        stem cell purging of immunoreactive cells, in preparation for        allogeneic or autologous stem cell transplantation.    -   v) Potential utilization of photosensitizers for ex vivo        elimination of reactive immune cells in patients with        immunological disorders.        The Rhodamine Dyes

Rhodamine 123 (2-(6-amino-3-imino-3H-xanthen-9-yl) benzoic acid methylester hydrochloride), a lipophilic cationic dye of the pyrylium classwhich can disrupt cellular homeostasis and be cytostatic or cytotoxicupon high concentration exposure and/or photodynamic therapy, althoughwith a very poor quantum yield (Darzynkiewicz Z., Carter S. (1988)Cancer Res., 48: 1295-1299). It has been used in vitro as a specificfluorescent stain for living mitochondria. It is taken up and ispreferentially retained by many tumor cell types, impairing theirproliferation and survival by altering membrane and mitochondrialfunction (Oseroff A. R. (1992) In Photodynamic therapy (Henderson B. W.,Dougherty T. J., eds) New York: Marcel Dekker, pp. 79-91). In vivo,chemotherapy with rhodamine 123 can prolong the survival of cancerousmice, but, despite initial attempts to utilize rhodamine 123 in thetreatment of tumors, the systemic toxicity of rhodamine 123 may limitthe usefulness (Bernal, S. D., et al. (1983) Science, 222: 169; Powers,S. K. et al. (1987) J. Neurosur., 67: 889).

U.S. Pat. No. 4,612,007 issued on Sep. 16, 1986 in the name of RichardL. Edelson, discloses a method for externally treating human blood, withthe objective of reducing the functioning lymphocyte population in theblood system of a human subject. The blood, withdrawn from the subject,is passed through an ultraviolet radiation field in the presence of adissolved photoactive agent capable of forming photoadducts withlymphocytic-DNA. This method presents the following disadvantages anddeficiencies. The procedure described is based on the utilization ofknown commercially available photoactive chemical agents for externallytreating patient's blood, leaving the bone marrow and potential residentleukemic clones intact in the process. According to Richard L. Edelson,the method only reduces, does not eradicate, the target cell population.Moreover, the wavelength range of UV radiation used in the processproposed by Richard L. Edelson could be damageable to the normal cells.

International Application published on Jan. 7, 1993 under Internationalpublication number WO 93/00005, discloses a method for inactivatingpathogens in a body fluid while minimizing the adverse effects caused bythe photosensitive agents. This method essentially consists of treatingthe cells in the presence of a photoactive agent under conditions thateffect the destruction of the pathogen, and of preventing the treatedcells from contacting additional extracellular protein for apredetermined period of time. This method is concerned with theeradication of infectious agents from collected blood and itscomponents, prior to storage or transfusion.

It would be highly desirable to be provided with new rhodaminederivatives for the treatment of immunereactive cells which overcomesthese drawbacks while having no systemic toxicity for the patient.

Halogenated rhodamine salts are dyes that have the property ofpenetrating cells and generally localising at the mitochondria. Theyhave been used in conjunction with photoactivation to kill certain typesof cells, namely cancer cells in Leukemia, and activated T-cells inautoimmune diseases.

The generally accepted mechanism for the cell killing effect is theproduction of singlet oxygen which is the reactive intermediate in thedisruption of the life-sustaining biological processes of the cell.

The role of the rhodamine dye in the production of singlet oxygen isthat of a photosensitizer, i.e. that of a molecule which absorbs theincident light energy and transfers it to ground state oxygen, therebyelevating it to its singlet excited state which is the reactiveintermediate.

It is further known that the efficiency of the energy transfer processis greatly enhanced by the presence of heavy atoms such as halogens onthe aromatic chromophore of the dye.

One critical problem that has not been addressed however is thedifferential uptake of the photosensitizer by the target cells relativeto the other, normal, cells. Indeed, it is known that uptake isgenerally a function of the molecular structure of the dye beingabsorbed and that this property varies with different cell types.

It would therefore be highly desirable to be provided with a series ofnew halogenated rhodamine dyes bearing a variety of substituents atdifferent positions of the molecule thereby making available newselective dyes for specific target cells.

One aim of the present invention is to produce new photosensitizersendowed with the following characteristics:

-   -   i) preferential localization and uptake by the immunoreactive        cells;    -   ii) upon application of appropriate light intensities, killing        those cells which have accumulated and retained the        photosensiting agents;    -   iii) sparing of the normal hemopoietic stem cell compartment        from the destructive effects of activated photosensitizers;    -   iv) potential utilization of photosensitizers for hematopoietic        stem cell purging of immunoreactive cells in preparation for        allogeneic or autologous stem cell transplantation; and    -   v) Potential utilization of photosensitizers for ex vivo        elimination of reactive immune cells in patients with        immunological disorders.

Therefore, in accordance with the present invention, there is provided aseries of new rhodamine derivatives alone or in association with apharmaceutically acceptable carrier; whereby photoactivation of saidderivatives induces cell killing while unactivated derivatives ofgeneral structure represented by the formula (I), and salts thereof, aresubstantially non-toxic to cells.

In accordance with the present invention, there is also provided withthe use of the photoactivable rhodamine derivatives according to theinvention for the photodynamic treatment for the selective destructionand/or inactivation of immunologically reactive cells without affectingthe normal cells and without causing systemic toxicity for the patient,wherein appropriate intracellular levels of said derivatives areachieved and irradiation of a suitable wavelength and intensity isapplied.

In accordance with the present invention, there is also provided amethod of prevention of graft-versus-host disease associated withallogeneic stem cell transplantation in a patient, which comprises thesteps of:

-   -   a) activating lymphocytes from a donor by mixing donor cells        with host cells for a time sufficient for a period of time        sufficient for an immune reaction to occur,    -   b) substantially eliminating the activated lymphocytes of        step a) with photodynamic therapy using a therapeutic amount of        a photoactivable derivative or composition of claim 1 under        irradiation of a suitable wavelength; and    -   c) performing allogenic stem cell transplantation using the        treated mix of step b).

In accordance with the present invention, there is provided a method forthe treatment of immunologic disorder in a patient, which comprises thesteps of:

-   -   a) harvesting said patient's hematopoietic cells;    -   b) ex vivo treating of the hematopoietic cells of step a) by        photodynamic therapy using a therapeutic amount of a        photoactivable derivative or composition of claim 1 under        irradiation of a suitable wavelength; and    -   c) performing graft infusion or autograft transplantation using        the treated hematopoietic cells of step b).

The immunologic disorder may be selected from the group consisting ofconditions in which self cells or donor cells react against host tissuesor foreign targets, such as graft-versus-host disease, graft rejection,autoimmune disorders and T-cell mediated immunoallergies.

The hematopoietic cells may be selected from the group consisting ofbone marrow, peripheral blood, and cord blood mononuclear cells.

For the purpose of the present invention the following terms are definedbelow.

The term “immunoreactive disorders” is intended to mean any alloimmuneor autoimmune reaction and/or disorders.

In accordance with other aspects of the present invention, theserhodamine compounds which are prepared following the general strategy ofhalogenating known and readily available rhodamine dyes therebygenerating a first and varied series of intermediates, which themselvescan serve as potential photosensitizers or, use these halogenatedrhodamines as intermediates in the synthesis of a second series ofrhodamine dyes whereby one or more halogen has been substituted for oneof the groups of structure (I). In the case where all of the halogensare replaced by new groups, a subsequent halogenation step is added tothe sequence to obtain the desired compound of structure I (see FIGS. 1to 5).

Testing of these compounds on various types of cells surprisinglyrevealed some of the candidate molecules to be non-toxic, more efficientand more selective than the known halogenated rhodamine dyes.

SUMMARY OF THE INVENTION

The present invention relates to rhodamine derivatives of the formula(I)

wherein:

-   -   one of R₁, R₂, R₃, R₄, and R₁₀ represents an halogen atom and        each of the remaining R₁, R₂, R₃, R₄, and each of the remaining        R₁₀ group is independently selected in the group constituted by        hydrogen, halogen atoms, an amino, acylamino, dialkylamino,        cycloalkylamino, azacycloalkyl, alkylcycloalkylamino,        aroylamino, diarylamino, arylalkylamino, aralkylamino,        alkylaralkylamino, arylaralkylamino, hydroxy, alkoxy, aryloxy,        aralkyloxy, mercapto, alkylthio, arylthio, aralkylthio,        carboxyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl,        carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, cyano,        hydroxysulfonyl, amidosulfonyl, dialkylamidosulfonyl,        arylalkylamidosulfonyl, formyl, acyl, aroyl, alkyl, alkylene,        alkenyl, aryl, aralkyl, vinyl, alkynyl group and by the        corresponding substituted groups;    -   m=0-1;    -   n=1-4;    -   A is nil, O, or NH;    -   R₉ represents an alkylene group;    -   Z is H, amino, dialkylamino, or trialkylamino salt;    -   X is an anion; and    -   R₅, R₆, R₇, and R₈ are independently H or C₁-C₆ alkyl or R₁ in        combination with R₅ or R₆, or R₂ in combination with R₅ or R₆,        or R₃ in combination with R₇ or R₈, or R₄ in combination with R₇        or R₈ represents an alkylene,        alone or in association with a pharmaceutically acceptable        carrier.

The invention also relates to intermediates of the formula (II) to (VII)and to those of formula (I′) as defined in 1 to 5, which are usefulinter alia in the synthesis of the rhodamine derivatives of formula (I).

The invention further relates to the new processes for the synthesis ofnew rhodamines derivatives of formula (I), wherein the various groups R₁to R₁₀, A, X, Y, Y′ and Z, and m and n are as previously defined,without the exclusion of the compounds listed in the proviso at the endof the previous definition. This processes being defined by the schemesrepresented in FIGS. 1 to 5 and by the corresponding parts of thedescription.

The rhodamine derivatives of the invention are useful alone or incombination with a carrier, for treating infections generated by Gram+and/or by Gram− bacteria. As well as in the treatment of diseasesgenerated by enveloped viruses or by non-enveloped viruses.

Those compounds are also useful in the in-vivo and ex-vivo treatment ofimmunologic disorders.

BRIEF DESCRIPTION OF THE SCHEMES

FIG. 1 is the general synthesis of substituted 4 and 2,7 halogenatedrhodamine derivatives.

FIG. 2 is the general synthesis of substituted 2 and 4,5 halogenatedrhodamine derivatives.

FIG. 3 is the general synthesis of substituted 4- and 2,7-halogenatedrhodamine derivatives.

FIG. 4 is the general synthesis of substituted 2- and 4,5-halogenatedrhodamine derivatives.

FIG. 5 is the general synthesis of substituted 2- and 4,5-halogenatedrhodamine derivatives.

FIG. 6 is the bacteriostatic activity of rhodamine derivatives againstE. coli; the bacterial strain E. coli was treated with the rhodaminederivatives at 50 uM without extrusion time. The determined effects areexpressed in log decrease of bacterial growth: HA-X-44: eradication;HA-X-164: 0.25 log; HA-X-171: 3.7 logs; HA-VIII-92: 6.2 logs; TH9402: 7logs. LB is growth without compounds.

FIG. 7 is bacteriostatic activity of rhodamine derivatives against P.aeruginosa; the bacterial strain P. aeruginosa was treated with therhodamine derivatives at 50 M without extrusion time. The determinedeffects are expressed in log decrease of bacterial growth: TH9402: 2logs. LB is growth without compounds.

FIG. 8 is bacteriostatic activity of rhodamine derivatives against S.typhimurium; the bacterial strain S. typhimurium was treated with therhodamine derivatives at 50 M without extrusion time. The determinedeffects are expressed in log decrease of bacterial growth: XA-X-44: 5logs; HA-X-164; 0.3 log; TH9402: 6.7 logs. LB is growth withoutcompounds.

FIG. 9 is bacteriostatic activity of rhodamine derivatives against P.aeruginosa; the bacterial strain P. aeruginosa was treated with therhodamine derivatives at 50 μM without extrusion time. The determinedeffects are expressed in log decrease of bacterial growth: TH9402: 2logs. LB is growth without compounds.

FIG. 10 is antiviral activity of rhodamine derivatives tested oncytomegalovirus; log decreases of viral infectivity and proliferation inFS cells. Compounds were added at 50 μM without extrusion time. Logdecreases of viral infectivity and proliferation in FS cells: compoundswere added at 50 μM without extrusion time and without light activation.

FIG. 11 is staphilococcus epidermitis; TH9402 inhibits bacterial growthof S. epidermitis at 50 μM without extrusion time.

FIG. 12 is staphilococcus epidermitis; HA-X-40 exhibits a bacteriostaticeffect on the growth of S. epidermitis with a 2 logs decrease ofbacterial growth at 50 IpM without extrusion time.

FIG. 13 is staphilococcus epidermitis; HA-X-40 eradicates bacterialgrowth of S. epidermitis at 50 μM with 90 minutes extrusion time.

FIG. 14 is, staphilococcus epidermitis; XA-X-44 eradicates bacterialgrowth of S. epidermitis at 50 μM without extrusion time.

FIG. 15 is staphilococcus epidermitis; HA-X-149 exhibits abacteriostatic effect on the growth of S. epidermitis with a 4.5 logsdecrease of bacterial growth at 50 M without extrusion time.

FIG. 16 is staphilococcus epidermitis; HA-X-164 exhibits abacteriostatic effect on the growth of S. epidermitis with a 3 logsdecrease of bacterial growth at 50 μM without extrusion time.

FIG. 17 is staphilococcus epidermitis; HA-X-171 exhibits abacteriostatic effect on the growth of S. epidermitis with a 6.5 logsdecrease of bacterial growth at 10 μM without extrusion time.

FIG. 18 is staphilococcus epidermitis; HA-VIII-92 exhibits abacteriostatic effect on the growth of S. epidermitis with a 4 logsdecrease of bacterial growth at 10 μM without extrusion time.

The following references mean:

-   HA-X-164: the acetate salt of 2,7-dibromorhodamine B methyl ester    (4)-   HA-X-149: the acetate salt of 2,7-dibromorhodamine B hexyl ester (8)-   HA-X-171: 4,5-dibromorhodamine 6G (11)-   HA-X-40: 2′-(6-dimethylamino-3-dimethylimino-3H-xanthen-9-yl) 4′,    5′-dichloro-benzoic acid methyl ester hydrochloride (10)-   HA-X-44: 4, 5-dibromorhodamine 110 2-(2-methoxy ethoxy) ethyl esther    (13)-   HA-VIII-92: rhodamine B 3-bromopropyl ester (14)-   TH 9402: 4, 5-dibromorhodamine methyl ester 123.

DETAILED DESCRIPTION OF THE INVENTION

A first object of the present invention is constituted by the newrhodamines derivatives of the formula (I)

wherein: (0)

-   -   one of R₁, R₂, R₃, R₄, and R₁₀ represents an halogen atom and        each of the remaining R₁, R₂, R₃, R₄, and each of the remaining        R₁₀ group is independently selected in the group constituted by        hydrogen, halogen atoms, an amino, acylamino, dialkylamino,        cycloalkylamino, azacycloalkyl, alkylcycloalkylamino,        aroylamino, diarylamino, arylalkylamino, aralkylamino,        alkylaralkylamino, arylaralkylamino, hydroxy, alkoxy, aryloxy,        aralkyloxy, mercapto, alkylthio, arylthio, aralkylthio,        carboxyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl,        carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, cyano,        hydroxysulfonyl, amidosulfonyl, dialkylamidosulfonyl,        arylalkylamidosulfonyl, formyl, acyl, aroyl, alkyl, alkylene,        alkenyl, aryl, aralkyl, vinyl, alkynyl group and by the        corresponding substituted groups;    -   m=0-1;    -   n=1-4    -   A is nil, O, or NH;    -   R_(Q) represents an alkylene group;    -   Z is H, amino, dialkylamino, or trialkylamino salt;    -   X is an anion; and    -   R₅, R₆, R₇, and R₈ are independently H or C₁-C₆ alkyl or R₁ in        combination with R₅ or R₆, or R₂ in combination with R₅ or R₆,        or R₃ in combination with R₇ or R₈, or R₄ in combination with R₇        or R₈ represents an alkylene,        alone or in association with a pharmaceutically acceptable        carrier,        with the proviso that the following specific compounds:    -   4,5-dibromorhodamine 123        (2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid        methyl ester hydrochloride) also called TH9402;    -   4,5-dibromorhodamine 123        (2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid        ethyl ester hydrochloride);    -   4,5-dibromorhodamine 123        (2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid        octyl ester hydrochloride);    -   4,5-dibromorhodamine 110 n-butyl ester        (2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid        n-butyl ester hydrochloride); and    -   rhodamine B n-butyl ester (2-(6-ethyl amino-3-ethyl        imino-3H-xanthen-9-yl)-benzoic acid n-butyl ester hydrochloride)        are excluded.

According to a preferred embodiment of this object of the invention

-   -   “alkyl” means a straight or branched aliphatic hydrocarbon group        and the corresponding substituted alkyl group bearing one or        more substituents which may be the same or different and which        are selected in the group constituted by halo, aryl, hydroxy,        alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy,        aralkylthio, and cycloalkyl and “branched” means that a lower        alkyl group such as methyl, ethyl or propyl is attached to a        linear alkyl chain, preferred alkyl groups include the “lower        alkyl” groups which are those alkyl groups having from about 1        to about 6 carbons., exemplary alkyl groups are methyl, ethyl,        isopropyl, hexyl, cyclohexylmethyl, methyl or ethyl groups are        more preferred;    -   “cycloalkyl” means a non-aromatic ring preferably composed from        4 to 10 carbon atoms, and the cyclic alkyl may be partially        unsaturated, preferred cyclic alkyl rings include cyclopentyl,        cyclohexyl, cycloheptyl, the cycloalkyl group may be optionally        substituted with an aryl group substituent, the cyclopentyl and        the cyclohexyl groups are preferred;    -   “alkenyl” means an alkyl group containing a carbon-carbon double        bond and having preferably from 2 to 5 carbon atoms in the        linear chain, exemplary groups include allyl vinyl;    -   “alkynyl” means an alkyl group containing a carbon-carbon triple        bond and having preferably from 2 to 5 carbon atoms in the        linear chain, exemplary groups include ethynyl, propargyl;    -   “acyl” means an aromatic carbocyclic radical or a substituted        carbocyclic radical containing preferably from 6 to 10 carbon        atoms, such as phenyl or naphtyl or phenyl or naphtyl        substituted by at least one of the substituents selected in the        group constituted by alkyl, alkenyl, alkynyl, aryl, aralkyl,        hydroxy, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro,        trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl,        aralkoxycarbonyl, acylamino, aroylamino, carbamoyl,        alkylcarbamoyl, dialkylcarbamoyl, alkylthio, arylthio, alkylene        or —NYY′ where Y and Y′ are independently hydrogen, alkyl, aryl,        or aralkyl;    -   “aralkyl” means a radical in which an aryl group is substituted        for an alkyl H atom, exemplary aralkyl group is benzyl;    -   “acyl” means an alkyl-CO— group in which the alkyl group is as        previously described, preferred acyl have an alkyl containing        from 1 to 3 carbon atoms in the alkyl group, exemplary groups        include acetyl, propanoyl, 2-methylpropanoyl, butanoyl or        palmitoyl;    -   “aroyl” means an aryl-CO-group in which the aryl group is as        previously described and preferably contains from 6 to 10 carbon        atoms in the ring, exemplary groups include benzoyl and 1- and        2-naphtoyl;    -   “alkoxy” means an alkyl-O— group in which the alkyl group is as        previously described, exemplary alkoxy groups include methoxy,        ethoxy, n-propoxy, i-propoxy, n-butoxy, and heptoxy;    -   “aryloxy” means an aryl-O— group in which the aryl group is as        previously described, exemplary aryloxy groups include phenoxy        and naphthoxy;    -   “alkylthio” means an alkyl-S-group in which the alkyl group is        as previously described, exemplary alkylthio groups include        methylthio, ethylthio, i-propylthio and heptylthio;    -   “arylthio” means an aryl-S-group in which the aryl group is as        previously described, exemplary arylthio groups include        phenylthio, naphthylthio;    -   “aralkyloxy” means an aralkyl-O— group in which the aralkyl        group is as previously described, exemplary aralkyloxy group is        benzyloxy;    -   “aralkylthio” means an aralkyl-S— group in which the aralkyl        group is as previously described, exemplary aralkylthio group is        benzylthio;    -   “dialkylamino” means an —NYY′ group wherein both Y and Y′ are        alkyl groups as previously described, exemplary alkylamino        include ethylamino, dimethylamino and diethylamino;    -   “alkoxycarbonyl” means an alkyl-O—CO— group wherein the alkyl        group is as previously described, exemplary alkoxycarbonyl        groups include methoxy- and ethoxy-carbonyl;    -   “aryloxycarbonyl” means an aryl-O—CO— group wherein the aryl        group is as previously described, exemplary aryloxycarbonyl        groups include phenoxy- and naphthoxy-carbonyl;    -   “aralkoxycarbonyl” means an aralkyl-O—CO— group wherein the        aralkyl is as previously defined, exemplary aralkoxycarbonyl        group is benzyloxycarbonyl;    -   “carbamoyl” is an H₂N—CO-group;    -   “alkylcarbamoyl” is an Y′YN—CO— group wherein one of Y and Y′ is        hydrogen and the other of Y and Y′ is alkyl as defined        previously;    -   “dialkylcarbamoyl” is an Y′YN—CO— group wherein both Y and Y′        are alkyl as defined previously;    -   “acylamino” is an acyl-NH group wherein acyl is as defined        previously;    -   “aroylamino” is an aroyl-NH group wherein aroyl is as defined        previously;    -   “akylene” means a straight or branched bivalent hydrocarbon        chain group having preferably from 2 to 8 carbon atoms, and the        alkylene group may be interrupted by one or more substituted        nitrogen atoms wherein the substituent of the nitrogen atom is        alkyl or oxygen or sulfur atoms, and it is presently more        preferred that the alkylene group has from 2 to 3 carbon atoms,        exemplary alkylene groups include ethylene (—CH₂CH₂—), propylene        (—CH₂CH₂CH₂—), —CH₂NMe-CH₂—, O—CH₂—O or —O—CH₂CH₂—O—;    -   “halo” preferably means fluoro, chloro, bromo or iodo;    -   “azacycloalkyl” preferably means a 4 to 9 membered saturated        carbon ring where one of the methylene groups is replaced by        nitrogen;    -   “cycloalkylamine” means an —NYY′ group wherein one of the Y and        Y′ is hydrogen and the other Y and Y′ is cycloalkyl as defined        previously;    -   “alkylcycloalkylamino” means an —NYY′ group wherein one of the Y        and Y′ is alkyl as defined previously and the other Y and Y′ is        cycloalky as defined previously;    -   “diarylamino” means an —NYY′ group wherein both Y and Y′ are        aryl groups as previously described;    -   “aralkylamino” means an —NYY′ group wherein one of the Y and Y′        is hydrogen and the other Y and Y′ is aralkyl as defined        previously;    -   “arylalkylamino” means an —NYY′ group wherein one of the Y and        Y′ is alkyl as defined previously and the other Y and Y′ is aryl        as defined previously;    -   “alkylaralkylamino” means an —NYY′ group wherein one of the Y        and Y′ is alkyl as defined previously and the other Y and Y′ is        aralkyl as defined previously;    -   “arylaralkylamino” means an —NYY′ group wherein one of the Y and        Y′ is aryl as defined previously and the other Y and Y′ is        aralkyl as defined previously;    -   “mercapto” is a —SH or a SR group wherein R may be any of the        above defined groups R₁ to R₁₀, the —SH, the mercaptoaryl and        the mercaptoalkyl groups are preferred;    -   “hydroxysulfonyl” is an —SO₃H;    -   “amidosulfonyl” is an —SO₂NH₂;    -   “dialkylamidosulfonyl” means an —SO₂NYY′ group wherein both Y        and Y′ are alkyl groups as previously described;    -   “arylaralkylamidosulfonyl” means an —SO₂NYY′ group wherein one        of the Y and Y′ is aryl as defined previously and the other Y        and Y′ is aralkyl as defined previously; and    -   “anion” means the deprotonated form of an organic or inorganic        acid and the anion is preferably selected from hydrochlorides,        hydrobromides, sulfates, nitrates, borates, phosphates,        oxalates, tartrates, maleates, citrates, acetates, ascorbates,        succinates, benzenesulfonates, methanesulfonates,        cyclohexanesulfonates, toluenesulfonates, sulfamates, lactates,        malonates, ethanesulfonates, cyclohexylsulfamates, and quinates.        In the case where the rhodamine derivative bears one or more        acidsubstituents, the covered compound comprise the internal        salt or any salt derived from neutralization by any of the        following bases: sodium hydroxide, potassium hydroxide, calcium        hydroxide, lithium hydroxide, ammonia, ethylene diamine, lysine,        diethanolamine, piperazine and the like.

A preferred embodiment of the invention is constituted by thoserhodamine derivatives wherein at least 2 of the R₁, R₂, R₃, R₄, and R₁₀groups represent an halogen atom which is preferably a bromide atom.

More preferred are those rhodamine derivatives, wherein the halogen(s)atom is(are) on the 2-7, 4-5 or 4′-5′ position on the ring or is(are) atthe end of the ester chain.

The following specific rhodamine derivatives are particularlyinteresting, the:

-   -   2′-(6-dimethylamino-3-dimethylimino-3H-xanthen-9-yl)        4′,5′-dichloro-benzoic acid methyl ester hydrochloride;    -   4,5-dibromo rhodamine 110 2-(2-methoxy ethoxy) ethyl ester;    -   acetate salt of 2,7-dibromorhodamine B hexyl ester;    -   acetate salt of 2,7-dibromorhodamine B methyl ester;    -   4,5-dibromorhodamine 6G; and    -   rhodamine B 3-bromopropyl ester.

A second object of the present invention is constituted by theintermediates represented by the formulae (II) to (VII) and (I′), theformulae being as defined in FIGS. 1 to 5, wherein the various groupsare as previously defined without any disclaimer. The intermediatesbeing as defined in FIGS. 1 to 5.

A third object of the present invention is constituted by new processesfor the synthesis of new rhodamines derivatives of formula (I) whereinthe various groups R₁ to R₁₀, A, X, Y, Y′ and Z, and m and n are aspreviously defined, without the exclusion of the compounds listed in theproviso at the end of the previous definition. These processes beingdefined by the schemes represented in FIGS. 1 to 5 and by thecorresponding parts of the description.

A fourth object of the present invention is constituted by the use of atleast one rhodamine derivatives as defined in the first object of theinvention, without the exclusion of the compounds listed in the provisoat the end of definition of the rhodamine derivative of formula (I),alone or in combination with a carrier, for treating infectionsgenerated by Gram+ and/or by Gram− bacteria.

According to a preferred embodiment of the present invention therhodamine derivatives are use for treating infections generated byStaphylococcus epidermitis.

Particularly interesting are:

-   -   the use of the 4,5-dibromo rhodamine 110 2-(2-methoxy ethoxy)        ethyl ester as bacteriostatic agent against Escherichia coli        0157:H7 and/or against Salmonella thyphimurium LT2;    -   the use of the acetate salt of 2,7-dibromorhodamine B hexyl        ester as bacteriostatic agent against Salmonella thyphimurium        LT2;    -   the use of the 4,5-dibromorhodamine 6G as bacteriostatic agent        against Escherichia coli 0157:H7;    -   the use of the rhodamine B 3-bromopropyl ester as bacteriostatic        agent against Escherichia coli 0157:H7; and    -   the use of the 4,5-dibromorhodamine methyl ester as        bacteriostatic agent against Escherichia coli 0157:H7,        Salmonella thyphimurium LT2 and/or Pseudomonas aeruginosa.

Preferably for this therapeutical use the rhodamine(s) derivative(s) is(are) combined with a carrier that is a pharmaceutically acceptablecarrier and is preferably selected in the group constituted by 5%mannitol and/or DMSO.

Any carrier is possible, however, the acceptable carrier is preferablyconstituted by 5% of mannitol: in water or in DMSO.

In the case of acetate salt of 2,7-dibromorhodamine B hexyl ester, ofthe HA-X-149, of the HA-X-164, the carrier is preferably constituted byDMSO.

A fifth object of the present invention is constituted by a bactericidalcomposition for the treatment of a liquid contaminated with Gram+ and/orGram− bacteria, which composition comprises an effective amount of atleast one rhodamine derivatives as above defined, without the exclusionof the compounds listed in the proviso at the end of claim 1, alone orin combination with a carrier.

A sixth object of the present invention is constituted by a bactericidalsolution, for the treatment of a locus contaminated with Gram+ and/orGram− bacteria, which solution comprises an effective amount of at leastone rhodamine derivatives of formula (I) as previously defined, withoutany disclaimer, alone or in combination with a carrier for treatinginfections generated by Gram+ and/or Gram− bacteria.

A seventh object of the present invention is constituted by a method fortreating infections generated by Gram+ and/or Gram− bacteria, whichmethod comprises administering to a human or animal in need an effectiveamount of at least one rhodamine derivatives of formula (I) aspreviously defined, without any disclaimer, alone or in combination witha carrier.

According to a preferred embodiment of this method, the effective amountadministered is comprises between 0.5 and 200 mg per kilogram bodyweight per day.

A eight embodiment of the present invention is constituted by amedicament containing an effective amount of at least one rhodaminederivatives of formula (I) as previously defined, without the exclusionof the compounds listed in the proviso at the end of the definition,alone or in combination with a carrier, for treating infectionsgenerated by Gram+ and/or Gram− bacteria.

A tenth object of the present invention is constituted by the use of aneffective amount of at least one rhodamine derivatives or salt thereofas above-defined without any disclaimer, alone or in combination with acarrier, in the treatment of diseases generated by enveloped viruses orby non-enveloped viruses.

Preferably, the enveloped virus is one with a double stranded ADN, morepreferably one of the Herpes viridae family.

An eleventh object of the present invention is constituted by medicamentcontaining an effective amount of at least one rhodamine derivatives orsalt thereof, as above, without the exclusion of the compounds listed atthe end of the definition of the rhodamine derivatives of formula (I),alone or in combination with a carrier, for treating viral infections.

Further preferred embodiment of the present invention are the use of:

-   -   the 4,5-dibromo rhodamine        2′-(6-dimethylamino-3-dimethylimino-3H-xanthen-9-yl)-4′,        5′-dichloro benzoic acid methyl ester hydrochloride;    -   4,5-dibromo rhodamine 110 2-(2-methoxy ethoxy) ethyl as an        antiviral agent against cytomegalovirus;    -   4,5-dibromorhodamine methyl ester as an antiviral agent against        cytomegalovirus; and    -   the acetate salt of 2,7-dibromorhodamine B hexyl ester        as an antiviral agent against Cytomegalovirus.

Another preferred embodiment of the invention is constituted by the2,7-dibromorhodamine B hexyl ester acetate salt as an antiviral agentagainst Cytomegalovirus.

Use of the acetate salt of 2,7-dibromorhodamine B hexyl ester as anantiviral agent against Cytomegalovirus

A twelfth object of the present invention is the use of the rhodaminederivatives of formula (I) as previously defined, without anydisclaimer, in the treatment of immunologic disorders.

According to a preferred embodiment, the use relates to enhancing highquantum-yield production and singlet oxygen generation upon irradiationwhile maintaining desirable differential retention of rhodamine betweennormal and cancer cells, said rhodamine derivatives of formula (I) beingas previously defined, without the disclaimer.

According to another embodiment, the use relates to the photodynamictherapy of cancer patients by destroying human cancer cells, whereinappropriate intracellular levels of said derivatives are achieved andirradiation of a suitable wavelength and intensity is applied.

According to a further preferred embodiment, the use of the inventionrelates to a method for the photodynamic therapy of patients sufferingfrom leukemias, disseminated multiple myelomas or lymphomas, whichcomprises the steps of:

-   -   a) harvesting said patient's human bone marrow;    -   b) purging of the bone marrow of step a) by photodynamic therapy        using a therapeutic amount of a photoactivable derivative        according to formula (I), without the exclusion of the compounds        listed in the proviso at the end of the definition, under        irradiation of a suitable wavelength; and    -   c) performing autologous stem cell transplantation using the        purged bone marrow of step b).

Preferably, the purging of step b) further comprises intensivechemotherapy and total body irradiation (TBI) procedures.

Another preferred embodiment relates to a method for in vitro purging ofthe human bone marrow containing metastasis of solid tumors, selectedfrom the group consisting of metastasis of breast, lung, prostatic,pancreatic and colonic carcinomas, disseminated melanomas and sarcomas,wherein surgical excision or debulking can be achieved, which comprisesthe steps of:

-   -   a) harvesting said patient's human bone marrow;    -   b) purging of the bone marrow of step a) by photodynamic therapy        using a therapeutic amount of at least one photoactivable        derivative of formula (I) as above defined without any        disclaimer, under irradiation of a suitable wavelength; and    -   c) performing autologous stem cell transplantation using the        purged bone marrow of step b).

Preferably, the purging of step b) further comprises intensivechemotherapy and total body irradiation (TBI) procedures.

A further embodiment of this object of the invention is a method for thephotodynamic therapy of cancer patients, which comprises administeringto said patients a therapeutically acceptable intracellular level of atleast one photoactivable derivative of formula (I) as above defined,without disclaimer, and subjecting said patients to irradiation of atherapeutically suitable wavelength.

Preferably, at least one photoactivable derivative is administered byinstillation, injection, bloodstream diffusion at the tumor sitesdirectly accessible to light emission or tumor sites accessible to laserbeams using rigid or flexible endoscopes.

More preferably, the laser-accessible tumor site is selected from thegroup consisting of urinary bladder, oral cavity, esophagus, stomach,lower digestive tract, upper and lower respiratory tract.

Another preferred embodiment of this object of the invention isconstituted by a method for the treatment of leukemias in patients,which comprises the steps of:

-   -   a) purging of cancerous clones from the bone marrow of said        patients;    -   b) subjecting said purged clones of step a) to a photodynamic        treatment using a therapeutical amount of at least one of the        photoactivable derivatives of formula (I) as previously defined,        without the disclaimer present at the end of the definition,        under irradiation of a suitable wavelength for the selective        destruction of leukemic cells without affecting the normal cells        of the patients; and    -   c) administering said treated clones of step b) to the patients;        thereby causing no systemic toxicity for the patients.

A fourteenth object of the present invention is constituted by aphotoactivable pharmaceutical composition for the selective destructionand/or inactivation of immunologically reactive cells without affectingthe normal cells and without causing systemic toxicity for the patient,said composition comprising at least one photoactivable rhodaminederivative of formula (I) as previously defined, without the exclusionof the compounds listed in the proviso at the end of the definition, andphotoactivable derivatives thereof; in association with apharmaceutically acceptable carrier; whereby photoactivation of saidderivatives induces cell killing while inactivated derivatives aresubstantially non-toxic to cells.

A fifteenth object of the present invention is constituted by the use ofthe photoactivable derivatives of claim 1 for the photodynamic treatmentfor the selective destruction and/or inactivation of immunologicallyreactive cells without affecting the normal cells and without causingsystemic toxicity for the patient, wherein appropriate intracellularlevels of said derivatives are achieved and irradiation of a suitablewavelength and intensity is applied.

-   -   A preferred embodiment is constituted by a method of prevention        of graft-versus-host disease associated with allogeneic stem        cell transplantation in a patient, which comprises the steps of:    -   a) activating lymphocytes from a donor by mixing donor cells        with host cells for a time sufficient for a period of time        sufficient for an immune reaction to occur;    -   b) substantially eliminating the activated lymphocytes of        step a) with photodynamic therapy using a therapeutic amount of        a photoactivable composition of claim 24 under irradiation of a        suitable wavelength; and    -   c) performing allogenic stem cell transplantation using the        treated mix of step b).

Another preferred embodiment is constituted by a method for thetreatment of immunologic disorder in a patient, which comprises thesteps of:

-   -   a) harvesting said patient's hematopoietic cells;    -   b) ex vivo treating of the hematopoietic cells of step a) by        photodynamic therapy using a therapeutic amount of a        photoactivable composition of claim 24 under irradiation of a        suitable wavelength; and    -   c) performing graft infusion or autograft transplantation using        the treated hematopoietic cells of step b).

Preferably, the immunologic disorder is selected from the groupconsisting of conditions in which self cells or donor cells reactagainst host tissues or foreign targets, such as graft-versus-hostdisease, graft rejection, autoimmune disorders and T-cell mediatedimmunoallergies.

More preferably, the hematopoietic cells is selected from the groupconsisting of bone marrow, peripheral blood, and cord blood mononuclearcells.

Compounds of structure I exhibit enhanced properties as: labeling dyesfor deoxynucleotides, dideoxynucleotides and polynucleotides; novel dyessuitable for recording fluids for the ink jet process; novel dyes forfiberglass and paper; novel dyes for the eradication of infectiousbiological contaminants in body tissues; novel dyes applicable inphotographic processes; novel dyes applicable in cancer chemotherapy;novel dyes applicable as inhibitors of the herpes simplex virusthymidine kinase and in the treatment and/or in the prophylaxis ofinfections caused the herpes simplex virus; novel dyes for use aspolymer optical amplifiers and lasers; novel dyes applicable in cellbiology; novel dyes applicable in the doping of siliceous materials togive solid dye lasers; novel pigments applicable for paints, inks andplastics; novel organic reagents in solvent extraction of metal ions;novel dyes applicable in the formation of new conjugate products withother dyes; novel dyes for the manufacture of CD-ROM type optical memorydisks; novel dyes applicable in the fluorophore labeling of peptides;novel dyes applicable in the flow cytometry analysis; novel dyesapplicable as stains for the detection of Mycobacterium tuberculosis byfluorescence microscopy; novel dyes applicable in the fluorescentmapping of binding sites for substrates, ligands and inhibitors, noveldyes to study transport through the blood-brain-barrier; novel dyes tostudy biofilm desinfection; novel dyes applicable as fluorescent probesin cell biology; novel dyes for use as water tracing; novel dyes forvisualization of peptide receptors by image intensified fluorescencemicroscopy; novel dyes for the formation of metal chelates in analyticalchemistry; novel fluorescent dyes applicable in diagnosis therapy.

Chemical Synthesis

These compounds are prepared following the general strategy ofhalogenating known and readily available rhodamine dyes therebygenerating a first and varied series of intermediates, which themselvescan serve as potential photosensitizers or, use these halogenatedrhodamines as intermediates in the synthesis of a second series ofrhodamine dyes whereby one or more halogen has been substituted for oneof the groups of structure I. In the case where all of the halogens arereplaced by new groups, a subsequent halogenation step is added to thesequence to obtain the desired compound of structure I, (see theillustrative schemes I and 2).

Due to the specific retention of the rhodamine 123 class of dyes by theabnormal malignant cells and the concomitant lack of their accumulationby the normal hematopoietic stem cells, these results provide evidencefor the potential use of these three new dyes for in vivo or in vitrophotodynamic therapy.

In accordance with the present invention, there is provided the use ofsuch above-mentioned dyes in conjugation with tumor specific antibodies,or poisonous substances, or liposomal or lipoproteins, or fluorochromeadducts.

In addition, the photosensitizers to be described have the potential toact synergistically in conjunction with other photoactive substances.

Moreover, the negative selection procedure provided by the use ofphotodynamic treatment does not preclude the use of other means forenriching hematopoietic stem cells such as positive selection withanti-CD34 monoclonal antibodies.

Other Clinical Applications

In addition to using photosensitizers in the context of in vitro bonemarrow purging for the leukemias and metastatic cancers, the moleculescan also be used in vivo for tumor sites directly accessible to exposureto a light source and to appropriate local concentrations of the drugsto be described. The molecules of the invention can also be utilized inthe photodynamic therapy of a patient suffering from disseminatedmultiple myelomas or lymphomas. The metastatic cancers for which thetherapy of this invention is appropriate include metastasis of breast,lung, prostatic, pancreatic and colonic carcinomas, disseminatedmelanomas and sarcomas. The photoactivable derivatives of the presentinvention can be administered by instillation, injection, bloodstreamdiffusion at the tumor sites directly accessible to light emission oftumor sites accessible to laser beams using rigid or flexibleendoscopes.

DESCRIPTION OF PREFERRED EMBODIMENTS

As a matter of illustration only, 5 methods of treatment of immunologicdisorders involving the rhodamine derivatives according to the inventionare thereafter illustrated.

Method I of Treatment of Leukemias

1. Diagnostic Procedures

Diagnosis of chronic myelogenous leukemia (CML) will be establishedusing one or more of the following procedures on blood or bone marrowcells:

-   -   a) conventional cytogenetics studies with identification of Ph        1+ metaphases harbouring the t(9:22);    -   b) fluorescent in situ hybridization for the detection of the        bcr/abl rearrangement; and    -   c) Southern blot analysis for the detection of a rearranged her        fragment or PCR-RT for the detection of chimeric ber/abl        messenger RNA.        2. Bone Marrow Harvesting

After diagnosis, bone marrow (BM) or peripheral blood (PB) derivedhemopoietic stem cells will be harvested using previously describedprocedures for the autologous marrow transplantation in cancer therapy(reviewed by Herzig G P, (1981) Prog. Hematol., 12:1). Hemopoietic stemcells collected for autograft will be immediately treated ex vivo asdescribed below.

3. In Vitro Purging of Leukemia

Ex vivo treatment consists, of short-term incubation or BM of PB stemcells with one or several of the selected photoactive compounds.Duration of incubation, cell concentration and drug molarity are bedetermined for each patient using an aliquot of the harvested cellpopulation. Excess of dyes will be removed by cell washes with steriledye free medium supplemented with 2% autologous serum. Cells are nextexposed to radiant energy of sufficient intensities to effectphotodynamic purging of leukemia cells. Efficacy of the photodynamicpurging procedure is verified on an aliquot of the treated cellpopulation, before cryopreservation and/or re-infusion to the patient isperformed. Until re-infusion to the patient, the cells are cryopreservedin 10% dimethyl sulfoxyde (DMSO)—90% autologous serum medium, at −196°C. in the vapour phase of liquid nitrogen.

4. Systemic Treatment of Patients

Following stem cell harvest, patient will be either treated withconventional regimens until autografting is clinically indicated orimmediately submitted to dose-intensive chemotherapy and total bodyirradiation where indicated.

5. Autologous Stem Cell Transplantation

Following appropriate treatment of the patient by high-dose chemotherapyand irradiation and at the appropriate clinical moment, cryopreservedmarrow or peripheral blood stem cells will be rapidly thawed and dilutedin medium containing 25 UI DNase ml⁻¹ to minimize clumping. A minimum of2×10⁷/kg nucleated cells with 85% to 95% viability as measured byTrypan™ blue exclusion will be returned to the patient.

Method II of Treatment of Malignancies

1. Diagnostic Procedures

Diagnosis of malignancies will be established using conventionalhistopathological examination of the primary tumor. Detection of marrowinvolvement by neoplastic cells will be achieved by direct histologicalexamination and ancillary procedures where indicated (i.e.immuno-peroxydase, immunohistochemical, tumor markers and hybridizationstudies).

2. Bone Marrow Harvesting

After diagnosis, bone marrow (BM) or peripheral blood (PB) derivedhemopoietic stem cells will be harvested using previously describedprocedures for the autologous marrow transplantation in cancer therapy(reviewed by Herzig G P, (1981) Prog. Hematol., 12:1). Hemopoietic stemcells collected for autograft will be treated immediately ex vivo asdescribed below.

3. In Vitro Purging of Leukemia

Ex vivo treatment will consist of short-term incubation of BM of PB stemcells with one or several of the selected photoactive compounds.Duration of incubation, cell concentration and drug molarity will bedetermined for each patient using an aliquot of the harvested cellpopulation. Excess of dyes will be removed by cell washes in sterile dyefree medium supplemented with 2% autologous serum. Cells will next beexposed to radiant energy of sufficient intensities to effectphotodynamic purging of leukemia cells. Whenever a sensitive molecularmarker is available, an aliquot of the treated cell population will betested for the detection of residual neoplastic cells beforecryopreservation and/or re-infusion to the patient is attempted. Thecells will be cryopreserved in 10% dimethyl sulfoxyde (DMSO)—90%autologous serum medium, at 196° C. in the vapour phase of liquidnitrogen.

4. Systemic Treatment of Patients

Following stem cell harvest, patient will be either treated withconventional regimens until autografting is clinically indicated orimmediately submitted to dose-intensive chemotherapy and total bodyirradiation where indicated.

5. Autologous Stem Cell Transplantation

Following high-dose chemotherapy and irradiation cryopreserved marrow orperipheral blood stem cells will be rapidly thawed and diluted in mediumcontaining 25 UI DNase M1⁻¹ to minimize clumping. A minimum of 2×10⁷/kgnucleated cells with 85% to 95% viability as measured by Trypan™ blueexclusion will be returned to the patient.

Method III of Prevention of Graft-Versus-Host Disease in the Context ofAllogeneic Stem Cell Transplantation

1. Diagnosis and Identification of Immunological Differences BetweenDonor and Recipient, and Graft-Versus-Host Disease:

Allogeneic stem cell transplantation is performed for numerousneoplastic and non-neoplastic conditions. Hematological malignancies arecomprised of leukemia, lymphoma, multiple myeloma, myelodysplasticsyndromes, etc.; and non-hematological malignancies: aplastic anemia,congenital disorders, severe immunodeficiency syndromes, rhumatoidarthritis, scleroderma, lupus erythematosus, multiple sclerosis, andother immune disorders.

Graft-versus-host disease is a complication of allogeneic stem celltransplantation, where donor cells react against host cells, damagingtarget tissues (usually skin, liver, gut, lung, lacrymal or salivaryglands, etc.). The diagnosis relies on several clinical and laboratoryparameters, that are extensively reviewed in Graft-vs.-Host Disease,Ferrara J L M, Deeg H J, Burakoff S J eds, Marcel Dekker, New York,1997.

GVHD develops against antigens present on recipient cells but not ondonor cells. Immunological differences between donor and recipient couldbe present at the level of major histocompatibility antigens, minorhistocompatibility antigens or tumor-associated antigens. Disparity willbe established using one or more of the following procedures on blood orbone marrow cells:

-   -   a) HLA typing: conventional serologic typing or molecular to        identify disparities between donor and recipient in major        histocompatibility complex class I and class II antigens; and    -   b) Mixed lymphocyte culture to identify differences in class II        antigens; and    -   c) Minor histocompatibility antigens: although a few cytotoxic T        cell lines are available and could be used to identify minor        histocompatibility antigens, currently, these tests are only        available for research purposes.        2. Progenitor Cell Harvesting

After diagnosis, bone marrow (BM) or peripheral blood (PB) or cord-bloodderived hemopoietic stem cells from the donor will be harvested usingpreviously described procedures for allogeneic progenitor celltransplantation (reviewed in Bone Marrow Transplantation, Forman S J,Blume K G, Thomas E D eds, Blackwell Scientific Publications, CambridgeMass., USA, 1994). Donor hemopoietic stem cells collected forallografting will be immediately incubated with irradiated (25Gy) hostmononuclear or other cells. Host cells admixed with donor cells areincubated in sterile dye free medium supplemented with 20% autologousserum and interleukin-2 for 2 days. This procedure elicits donor cellalloreactivity towards the host, and the cell graft subsequentlyundergoes photodynamic treatment ex vivo as described below.

3. Selective In Vitro Purging of Immunoreactive Cells

Ex vivo treatment will consist of short-term incubation of previouslyactivated BM or PB stem cells with one or several of the selectedphotoactive compounds. Duration of incubation, cell concentration anddrug molarity will be determined for each patient using an aliquot ofthe harvested cell population. Excess of dyes will be removed by cellwashes with sterile dye free medium supplemented with 2% autologousserum. Cells will next be exposed to radiant energy of sufficientintensities to effect photodynamic purging of leukemia cells. Efficacyof the photodynamic purging procedure will be verified on an aliquot ofthe treated cell population, before cryopreservation and/or re-infusionto the patient is performed. Until re-infusion to the patient, the cellswill be cryopreserved in 10% dimethylsulfoxyde (DMSO)—90% autologousserum medium, at −196° C. in the vapor phase of liquid nitrogen.

4. Systemic Treatment of Patients

Following stem cell harvest, the patient will be submitted todose-intensive chemotherapy and/or irradiation when indicated.

5. Allogeneic Stem Cell Transplantation

Following appropriate treatment of the patient by high-dose chemotherapyand/or irradiation and at the appropriate clinical moment, cryopreservedmarrow or peripheral blood or cord blood stem cells will be rapidlythawed and returned to the patient.

Method IV of Treatment of Graft-Versus-Host Disease and AutoimmuneDiseases

1. Diagnostic Procedures

Diagnosis of graft-versus-host disease or immunoreactive disorders willbe established using conventional clinical, biochemical and/orhistopathological examination of the blood or appropriate tissues.Diagnostic and predictive features of GVHD are reviewed inGraft-vs.-Host Disease, Ferrara J L M, Deeg H J, Burakoff S J eds,Marcel Dekker, New York, 1997.

2. Harvesting of Peripheral Blood Cells

After diagnosis of severe GVHD, autoimmune or immunoreactive disorder,peripheral blood (PB) mononuclear cells will be harvested usingpreviously described or similar leukopheresis procedures (reviewed inBone Marrow Transplantation, Forman S J, Blume K G, Thomas E D eds,Blackwell Scientific Publications, Cambridge Mass., USA, 1994).Patient's peripheral blood mononuclear cells collected will be treatedimmediately ex vivo as described below.

3. In Vitro Elimination of Cells Mediating GVHD

Ex vivo treatment will consist of short-term incubation of PB stem cellswith one or several of the selected photoactive compounds. Duration ofincubation, cell concentration and drug molarity will be determined foreach patient using an aliquot of the harvested cell population. Excessof dyes will be removed by cell washes in sterile dye free mediumsupplemented with 2% autologous serum. Cells will next be exposed toradiant energy of sufficient intensities to effect photodynamic purgingof activated cells, which mediate GVHD.

4. Administration of Photodynamically Treated Cells to Patients

Leukopheresed cells that are photodynamically treated will be reinfusedinto the patient. This approach will enable the elimination of a largenumber of circulating activated lymphocytes and other cells involved inGVHD. In addition, cells spared by the photodynamic treatment areunactivated and their reinfusion into the patient may help restorenormal immunologic equilibrium.

Method V of Treatment of Immunologic Disorders

1. Diagnostic Procedures

Diagnosis of autoimmune disorders will be established using conventionalclinical, biochemical and/or histopathological examination of the bloodor appropriate tissues. Severe autoimmune diseases are amenable toautologous transplantation (reviewed in Sullivan K M et al., Am. Soc.Hematol., Educ. Program Book, 1998: 198-214).

2. Harvesting of Hematopoletic Stem Cells

After diagnosis, bone marrow (BM), peripheral blood (PB) or cord blood(CB) mononuclear cells will be harvested using previously describedprocedures for the autologous marrow transplantation in cancer therapy(reviewed in Bone Marrow Transplantation, Forman S J, Blume K G, ThomasE D eds, Blackwell Scientific Publications, Cambridge Mass., USA, 1994).Patient's hemopoietic stem cells collected for autograft will be treatedimmediately ex vivo as described below.

3. In Vitro Elimination of Cells Mediating Autoimmune Disorders

Ex vivo treatment will consist of short-term incubation of BM or PB stemcells with one or several of the selected photoactive compounds.Duration of incubation, cell concentration and drug molarity will bedetermined for each patient using an aliquot of the harvested cellpopulation. Excess of dyes will be removed by cell washes in sterile dyefree medium supplemented with 2% autologous serum. Cells will next beexposed to radiant energy of sufficient intensities to effectphotodynamic purging of immunoreactive cells, which mediate theimmunologic disorder.

4. Administration of Photodynamically Treated Cells to Patients

Hematopoietic stem cells that are photodynamically treated will bestored (frozen or kept in culture). This approach will enable theelimination of a large number of activated lymphocytes and other cellsinvolved in the immunologic disorder. In addition, cells spared by thephotodynamic treatment are unactivated and their reinfusion may helprestore normal immunologic equilibrium. Following stem cell harvest,patient will be either treated with conventional regimens untilautografting is clinically indicated or immediately submitted todose-intensive chemotherapy and total body irradiation where indicated.

5. Autologous Stem Cell Transplantation

Following high-dose chemotherapy and irradiation cryopreserved marrow orperipheral blood stem cells will be rapidly thawed and infused to thepatient.

The preparation of those rhodamine derivatives of formula (I), as abovedefined, without the proviso, will be more readily understood byreferring to the following examples which are given for illustrativepurpose.

I Synthesis of 2,7-Dibromorhodamine B Methyl Ester Acetate Salt (4) I-1Preparation of Rhodamine B Methyl Ester (1)

To a stirred mixture of 1.63 g (3.40 mmol) of Rhodamine B and 100 ml ofmethanol, hydrochloric acid was bubbled through the solution for 45 minand the reaction mixture was refluxed overnight. The methanol wasevaporated under reduced pressure and the dark red residue was thenpurified by flash chromatography using a mixture of methanol anddichloromethane (1:9) as eluent to afford the desired product as a deepred viscous residue (1.54 g).

Rf: 0.52 (MeOH: CH₂Cl₂ 1.5: 8.5)

Yield: 92%

Ms (FAB): Calculated for C₂₉H₃₃O₃N₂: (M-Cl)⁺: 457.2491

-   -   Found (M-Cl)⁺: 457.2494

UV(MeOH): λ_(max) 555 nm

I-2 Preparation of Dihydrorhodamine B Methyl Ester (2)

Rhodamine B methyl ester 1.73 g (3.50 mmol) was dissolved in 250 ml ofdichromethane and 100 ml of water. Excess NaBH₄ (solid) was added inportion with vigorous stirring, during 30 min, until the initial darkred colour was discharged. The pale orange organic phase was separatedand the aqueous phase extracted twice with dicholoromethane. Thecombined organic layers were dried on Na₂SO₄, filtered and evaporatedunder reduced pressure and the residue purified by flash chromatographyusing ethyl acetate as the eluting solvent. Fractions containing theproduct were combined and the solvent evaporated to afford the product 2as a pink oil (1.50 g).

Rf: 0.84 (AcOEt)

Yield: 93.7%

I-3 Bromination of Dihydrorhodamine B Methyl Ester (2)

In a 250 ml round bottom flask we introduced dihydrorhodamine B methylester (2) 1.34 g (2.92 mmol) and 112 ml of methanol spectrograde. Themixture was stirred at room temperature until all the ester wasdissolved. Propylene oxide 2 eq. (409 μL, 5.85 mmol) was added followedby dropwise addition of bromine 2 eq. (300 μL, 5.85 mmol). The stirringwas continued at room temperature for 1 h 30 min. The volatile solventwere evaporated under reduced pressure and the red oily residue wassubjected to purification by flash chromatography using ethyl acetateand hexanes (0.5: 9.5) as eluent to give the desired compound 3 as foamwhite solid (570 mg)

Rf: 0.41 (AcOEt: Hexanes 0.5: 9.5)

Yield: 31.6%

Nmr: (CD₃OD) δ 7.86 (dd, J=1.44 and 7.8 Hz, 1H); 7.44 (m, 1H); 7.32 (m,1H); 7.16 (s, 2H); 7.10 (dd, J=1.45 and 7.8 Hz, 1H); 6.93 (s, 2H); 6.17(s, 1H); 3.94 (s, 3H); 3.09 (q, J=7.09 Hz, 8H); 1.04 (t, J=7.09 Hz,12H).

Ms (FAB): (MH)⁺ 615.1

I-4 Oxydation of the 2,7-dibromodlhydrorhodamine B Methyl Ester andFormation of the Acetate Salt of 2,7-dibromorhodamine B Methyl Ester (4)

To a stirred solution of 2,7-dibromodihydrorhodamine B methyl ester (3)400 mg (0.64 mmol) in 10 ml of dichloromethane was added chloranil (1.2eq., 0.77 mmol, 192 mg). The reaction mixture was stirred at roomtemperature overnight, then the reaction was stopped and the solvent wasevaporated under reduced pressure to give a purple residue. The oxidizedcompound obtained in the precedent step was dissolved in 15 ml ofdichloromethane and acetic acid (0.8 ml) was added dropwise. The clearred solution obtained was stirred for 5 min, at room temperature,followed by the evaporation of the volatile solvent under reducedpressure to give a purple viscous residue. The residue was purified byflash chromatography using a 10% methanol in dichloromethane as eluentto give the desired compound 4 as a viscous purple solid (200 mg).

Rf: 0.29 (MeOH: CH₂Cl₂ 1:9)

Yield: 45.7%

Nmr: (CD₃OD) δ 8.48 (dd, J=1.45 and 7.5 Hz, I H); 7.95 (m, 2H); 7.52(dd, J=1.6 and 7.2 Hz, 1H); 7.45 (s, 2H); 7.38 (s, 2H); 3.79 (q, J=8 Hz,81-1); 3.71 (s, 3H); 1.99 (s, 3H); 1.37 (t, J=7.02 Hz, 2H)

Ms (FAB): Calculated for C₂₉H₃₂O₃N₂Br₂ (MH-AcO)⁺ 614.0779

-   -   Found: 614.0765

UV (MeOH): λ_(max) 577 nm

Example II II Synthesis of 2,7-dibromorhodamine B Hexyl Ester AcetateSalt (8) II-1 Preparation of Rhodamine B Hexyl Ester (5)

To a stirred mixture of 2.39 g (4.98 mmol) of Rhodamine B and 120 ml of1-hexanol, hydrochloric acid was bubbled through the solution for 45 minand the reaction mixture was refluxed overnight. The I-hexanol was thendistilled under reduced pressure and the dark red residue was purifiedby flash chromatography using a mixture of methanol and dichloromethane(1:9) as eluent. After the evaporation of the volatile solvents weobtained a viscous red green residue (2.62 g).

Rf: 0.45 (MeOH: CH₂Cl₂ 1.2:8.8)

Yield: 93.5%

Ms (FAB): Calculated C₃₄H₄₃O₃N₂ (M-Cl)⁺: 527.3273

-   -   Found: 527.3261

UV (MeOH): λ_(max) 555 nm

II-2 Preparation of Dihydrorhodamine B Hexyl Ester (6)

Rhodamine B hexyl ester (5) 940 mg (1.66 mmol) was dissolved in 200 mlof dichromethane and 150 ml of water. Excess NaBH4 (solid) was added inportion with vigorous stirring, during 30 min, until the initial darkred colour was discharged. The pale orange organic phase was separatedand the aqueous phase extracted twice with dicholoromethane. Thecombined organic layers were dried on Na₂SO₄, filtered and evaporatedunder reduced pressure. The crude oil residue was purified by flashchromatography using ethyl acetate as eluent giving 794 mg of 6 as apinkish oil.

Rf: 0.92 (AcOEt)

Yield: 90%

II-3 Bromination of Dihydrorhodamine B Hexyl Ester (6)

In a 100 ml round bottom flask we introduced dihydrorhodamine B hexylester (6) 784 mg (1.48 mmol) and 25 ml of methanol spectrograde. Themixture was stirred at room temperature until all the ester wasdissolved. Propylene oxide 2 eq. (208 μL, 2.96 mmol) was added followedby dropwise addition of bromine 2 eq. (152 SL, 2.96 mmol). The stirringwas continued at room temperature for 1 h 30 min. The volatile solventwere evaporated under reduced pressure and the red oily residue wassubjected to purification by flash chromatography using ethyl acetateand hexanes (0.25: 9.75) as eluent to afford 207 mg of pure compound 2as white foam solid and 123 mg of impur product.

Rf: 0.61 (AcOEt: Hexanes 0.5: 9.5)

Yield: 20.5%

II-4 Oxydation of the 27-dibromodihydrorhodamine B Hexyl Ester andFormation of the Acetate Salt of 2,7-dibromorhodamine B Hexyl Ester (8)

To a stirred solution of 2,7-dibromo dihydro rhodamine B hexyl ester 207mg (0.30 mmol) in 8 ml of dichloromethane was added chloranil (1.2 eq.,0.36 mmol, 89 mg). The reaction mixture was stirred at room temperatureovernight, then the reaction was stopped and the solvent was evaporatedunder reduced pressure to give a purple residue. The oxidized compoundobtained in the precedent step was dissolved in 8 ml of dichloromethaneand acetic acid (0.8 ml) was added dropwise. The clear red solutionobtained was stirred for 5 min at room temperature followed by theevaporation of the volatile solvent under reduced pressure to give apurple viscous residue, which is purified by flash chromatography usinga 10% methanol in dichloromethane as eluent to give the desired compound8 as a viscous purple solid (198 mg).

Rf: 0.47 (MeOH: CH₂Cl₂ 1:9)

Yield: 86.9%

Nmr: (CD₃OD) δ 8.29 (dd, J=1.5 and 7.6 Hz, 1H); 7.82 (m, 2H); 7.40 (dd,J=1.6 and 7.2 Hz, I H); 7.37 (s, 2H); 7.28 (s, 2H); 3.96 (t, J=7.2 Hz,2H);

-   -   3.72 (q, J=7.05 Hz, 8H); 1.91 (s, 3H); 1.29 (t, J=7.06 Hz, 12H);        1.08 (m, 4H); 0.79 (t, J=7.04 Hz, 3H)

Ms (FAB): Calculated for C₃₄H₄₂O₃N₂Br₂ (MH-AcO)⁺: 684.1561

-   -   Found: 684.1587

UV (MeOH): λ_(max) 582 nm

Example III III Synthesis of2′-(6-dimethylamino-3-dimethylimino-3H-xanthen-9-yl)4′,5′-dichloro-benzoic Acid Methyl Ester Hydrochloride (10) III-1Preparation of 2′-(6-dimethylamino-3-dimethylimino-3H-xanthen-9-yl)4′,5′-dichloro-benzoic Acid Hydrochloride (9)

A mixture of 3.00 g (21.8 mmol) of 3-(dimethylamino) phenol, 3.00 g(13.8 mmol) of 4,5-dichlorophtalic anhydride, and 1.72 g of zincchloride is heated in an oil bath at 165-170° C. for 5 h 30 min withstirring. The melt is cooled and powdered to give a red solid. The solidis washed with hot water, triturated with 10% sodium hydroxide anddiluted with water. The gum which separates is collected, washed withmore sodium hydroxide and water. The resulting dye base is thentriturated with concentrated hydrochloride acid. Water was then addedand the red precipitate obtained was collected and dried. The dye wasthen dissolved in methanol and precipitated with diethyl ether to give 9as red solid (3.27 g).

Rf: 0.48 (MeOH: CH₂Cl₂ 2:8)

Yield: 48%

Nmr: (CD₃OD) δ 8.47 (s, 1H); 7.72 (s, 1H); 7.22 (d, J=9.47 Hz, 2H); 7.11(m, 2H); 7.01 (d, J=2.4 Hz, 2H); 3.32 (s, 12H)

Ms (FAB): Calculated for C₂₄H₂₁O₃N₂Cl₂ (M-Cl)⁺: 455.0929

-   -   Found: 455.0938

UV (MeOH): λ_(max) 511 nm

III-2 Preparation of2′-(6-dimethylamine-3-dimethylimino-3H-xanthen-9-yl) 4′.5-dichloro-benzoic Acid Methyl Ester Hydrochloride (10)

To a 250 ml round bottom flask, equipped with a magnetic stirrer, wasadded 738 mg (1.50 mmol) of the acid 9 and 40 ml of anhydrousdichloromethane and 10 ml of anhydrous DMF. The mixture was stirredunder nitrogen until all the acid was dissolved. An amount of 309 mg(1.50 mmol) of 1,3-dicyclohexylcarbodiimide (DCC) was then addedfollowed by 200 μL of methanol and 18 mg of 4-N,N-dimethylamino pyridine(DMAP). The mixture was stirred at room temperature overnight. Thesolvent was then distilled under reduced pressure to give a red residue,which was purified by flash chromatography using MeOH: CH₂Cl₂ (1.2:8.8)as eluant to afford 10 as red brown solid (350 mg).

Rf: 0.52 (MeOH: CH₂Cl₂ 2:8)

Yield: 46%

Nmr: (CD₃OD) δ 8.50 (s, 1H); 7.80 (s, 1H); 7.18 (d, J=9.2 Hz, 2H); 7.12(m, 2H); 7.04 (d, J=2.31 Hz, 2H); 3.80 (s, 3H); 3.35 (s, 12H)

Ms (FAB): Calculated for C₂₅H₂₃O₃N₂Cl₂ (M-Cl)⁺: 469.1085

-   -   Found: 469.1078

UV (MeOH): 555 nm

Example IV IV Preparation of 45-dibromorhodamine 6G (11)

To a quantity of 600 mg (1.25 mmol) of rhodamine 6G dissolved in 50 mlof methanol was added dropwise, at room temperature, a solution of 128 L(2 eq., 2.50 mmol) of bromine. A precipitate was formed 10 min after theaddition of the bromine. The mixture was stirred for 3 hours, and thesolvent was evaporated under reduced pressure to give a red solid. Thecrude was recrystallized from methanol: diethyl ether (80 ml: 400 ml) togive the product 11 as green red solid (585 mg).

Rf: 0.26 (MeOH: CH₂Cl₂ 1:9)

Yield: 68.5%

Nmr (CD₃OD): δ 8.36 (dd, J=1.13 and 7.44 Hz, 1H); 7.89 (m, 2H); 7.47(dd, J=1.46 and 6.76 Hz, I H); 6.98 (s, 2H); 4.07 (m, 6H); 2.29 (s, 6H);1.28 (t, J=7.04 Hz, 6H); 1.02 (t, J=7.05 Hz, 3H)

Ms (FAB): Calculated for C₂₈H₃₀O₃N₂Br₂ (MH-Br)⁺: 600.0623

-   -   Found: 600.0605

UV (MeOH): λ_(max) 546 nm

Example V V Synthesis of 4,5-dibromorhodamine 110 2-(2-methoxy Ethoxy)Ethyl Ester (13) V-I Preparation of Rhodamine 110 2-(2-methoxy Ethoxy)Ethyl Ester (12)

To Rhodamine 110 1.00 g (2.72 mmol) was added a mixture of anhydrous DMFand dichloromethane (60 ml: 10 ml) and the mixture was stirred until allthe dye was dissolved. 1,3-dicyclohexylcarbodiimide (DCC) 562 mg (1 eq.,2.72 mmol) was added followed by HOBT 368 mg (1 eq., 2.72 mmol),2-(2-methoxy ethoxy) ethanol 518 μL (1.60 eq., 4.36 mmol) and 33 mg(0.27 mmol) of 4-dimethylamino pyridine (DMAP). The reaction was stirredat room temperature overnight, and DMF was then distilled under reducedpressure to give a deep red residue. This residue was subjected topurification by flash chromatography using methanol: dichloromethane(2:8) as eluent to give (530 mg) of a red solid. Thin layerchromatography (TLC) showed the presence of another product with thedesired one. The solid obtained was then dissolved in methanol (10 ml)and diethyl ether was added until a precipitate was obtained. Theproduct was collected and dried to give the desired compound 12 (220 mg)as a red solid.

Rf: 0.33 (MeOH: CH₂Cl₂ 2:8)

Yield: 18.4%

Ms (FAB): Calculated for C₂₅H₂₅N₂O₅ (M-Cl)⁺: 433.1736

Found: 433.1777

V-2 Preparation of 4, 5-dibromo Rhodamine 110 2-(2-methoxy Ethoxy) EthylEster 13

To a 100 ml round bottom flask, equipped with a magnetic stirrer, wasadded 235 mg (0.50 mmol) of the rhodamine 110 2-(2-methoxy ethoxy) ethylester 12 and 15 ml of methanol spectrograde. The mixture was stirreduntil all the rhodamine dye was dissolved. An amount of 50 μL (2 eq.,1.00 mmol) of bromine was then added, and the reaction was stirred atroom temperature for 1 h 30 min. At the end of the reaction 10 μL ofcyclohexene was added and the mixture was stirred for another 10 min.The volatile solvent was evaporated under reduced pressure to give a redsolid. This solid was chromatographed on silica gel using MeOH:CH₂Cl₂(1.2:8.8) as eluting solvent.

The pure fractions were combined and evaporated to give compound 13 (250mg) as red solid.

Rf: 0.76 (MeOH: CH₂Cl₂ 2:8)

Yield: 74.3%

Nmr (CD₃OD): δ 8.38 (dd, J=1.5 and 6.87 Hz, 1H); 7.88 (m, 2H); 7.47 (dd,J=1.48 and 7.02 Hz, 1H); 7.15 (d, J=9.22 Hz, 2H); 7.04 (d, J=9.21 Hz,2H);

4.15 (m, 2H); 3.39-3.25 (m, 9H)

Ms (FAB): Calculated for C₂₅H₂₃O₅N₂Br₂ (M-Br)⁺: 588.9973

-   -   Found: 588.9962

UV (MeOH): λ_(max) 502 nm

Example VI VI Preparation of Rhodamine B 3-bromopropyl Ester (14)

To Rhodamine B 300 mg (0.62 mmol) was added 5 ml of dichloromethane andthe mixture was stirred until all the dye was dissolved. An amount of1,3-dicyclohexylcarbodiimide (DCC) 142 mg (1 eq., 0.62 mmol) was addedfollowed by 139 mg (10.0 mmol) of 3-bromopropanol and 8 mg (0.06 mmol)of 4-dimethyl aminopyridine (DMAP). The reaction was stirred at roomtemperature overnight. The N,N-dicyclohexyl urea was filtered and thesolvent evaporated in vacuuo to give a deep red residue which wassubjected to purification on flash chromatography using methanol:dichloromethane (1:9) as eluent. The fractions containing the desiredcompound were combined and and the solvent evaporated under reducedpressure to give 14 as a deep red viscous solid (300 mg)

Rf: 0.71 (MeOH: CH₂Cl₂ 1.5: 8.5)

Yield: 79.8%

Nmr (CD₃OD): δ 8.29 (m, I H); 7.85 (m, 2H); 7.43 (m, 1H); 7.06 (m, 6H);4.08 (m, 2H); 3.68 (q, J=7.06 Hz, 8H); 3.21 (m, 2H); 1.81 (m, 1H); 1.29(t, J=7.08 Hz, 12H)

Ms (FAB): Calculated for C₃₁H₃₆O₃N₂Br₁ (M-Cl)⁺: 563.1909

-   -   Found: 563.1921

UV (MeOH): λ_(max) 545 nm

Example VII VII Synthesis of 2,7-dibromo-4′-carboxytetramethylrosamineMethyl Ester Acetate Salt (18) VII-1 Preparation of4′-carboxydihydrotetrametylrosamine Methyl

Ester 15 910 mg (2.08 mmol) was dissolved in 250 ml of dichromethane and150 ml of water. Excess NaBH₄ (solid) was added in portion with vigorousstirring, during 30 min, until almost all color was discharged. The paleorange organic phase was separated and the water phase extracted twicewith dicholoromethane. The combined organic layers were dried on Na₂SO₄,filtered and evaporated under reduced pressure. The crude oil residuewas purified by flash chromatography using ethyl acetate as eluent,giving 530 mg of white foam solid.

Rf: 0.83 (AcOEt)

Yield: 63%

VII-2 Bromination of dihydro-4′-carboxytetramethylrosamine Methyl Ester(16)

In a 100 ml round bottom flask we introduced dihydro rhodamine B hexylester 530 mg (1.31 mmol) and 50 ml of methanol spectrograde. The mixturewas stirred at room temperature until all the ester was dissolved.Propylene oxide 2 eq. (185 μL, 2.63 mmol) was added followed by dropwiseaddition of bromine 2 eq. (135 μL, 2.63 mmol). The stirring wascontinued at room temperature for 1 h 30 min. The volatile solvent wereevaporated under reduced pressure and the red oily residue was subjectedto purification on flash chromatography using ethyl acetate and hexanes(1:9) as eluent to give a white foam solid (391 mg).

Rf: 0.36 (AcOEt:Hexanes 1:9)

Yield: 53.5%

Nmr (CD₃OD): δ 7.96 (d, J=8.5 Hz, 2H); 7.28 (d, J=8.31 Hz, 2H); 7.22 (s,2H);

-   -   6.94 (s, 2H); 3.87 (s, 3H); 2.77 (s, 12H)

VII-3 Oxydation of the 2,7-dibromodihydro-4′-carbomexytetramethylRosamine Methyl Ester (17) and Formation of the Acetate Salt of 2,7Dibromo-4′-Carboxytetramethylrosamine Methyl Ester (18)

To a stirred solution of 2, 7-dibromodiliydrotetramethylrhodamine methylester 390 mg (0.69 mmol) in 15 ml of dichloromethane was added chloranil(1.2 eq., 0.83 mmol, 205 mg). The reaction mixture was stirred at roomtemperature overnight, then the reaction was stopped and the solvent wasevaporated under reduced pressure to give a purple residue. The oxidizedcompound obtained was dissolved in 15 ml of dichloromethane and aceticacid (0.8 ml) was added dropwise which. The clear purple solutionobtained was stirred for 5 min at room temperature followed by theevaporation of the volatile under reduced pressure to give a purpleviscous residue, which is purified by flash chromatography using a 10%methanol in dichloromethane as eluent to give the desired compound 18Awhich is in equilibrium with compound 18B.

18A Rf: 0.34 (MeOH: CH₂Cl₂ 1:9)

18B Rf: 0.93 (MeOH: CH₂Cl₂ 1:9)

Yield: 30%

Nmr (CD₃OD): δ 7.97 (d, J=8.28 Hz, 2H); 7.45 (d, J=8.33 Hz, 2H); 7.19(s, 2H); 6.99 (s, 2H); 3.89 (s, 3H); 2.93 (s, 2, 64H); 2.83 (s, 12H);2.01 (s, 0, 356H)

Ms (FAB): Calculated for C₂₅H₂₄O₃N₂Br₂ (MH-AcO)⁺: 558.0153

-   -   Found: 558.0169

Example VIII Preparation of 4, 5-dibromo Rhodamine B Lactone (19)

Rhodamine B 500 mg (1.04 mmol) was dissolved in 25 ml of acetic acid and25 ml of water. Bromine 107 μL (2 eq., 2.08 mmol) was then addeddropwise and the reaction mixture was then stirred at room temperatureovernight. The water and the acetic acid were evaporated under reducedpressure and the residue obtained was redissolved in dichloromethane and10% aqueous solution of sodium bicarbonate.

The organic layer was separated and washed twice with water, dried onNa₂SO₄, filtered and evaporated to give a pink oil. The residue waschromatographed on silica gel using methanol: dichloromethane (0.2:9.8)as eluent to give 544 mg of white foam solid.

Rf: 0.88 (MeOH: CH₂Cl₂ 1:9)

Yield: 86.8%

Nmr (CD₃OD) S 7.89 (dd, J=1.45 and 7.8 Hz, 1H); 7.62 (m, 2H); 7.14 (dd,J=1.6 and 7.2 Hz, 1H); 6.81 (d, J=9.2 Hz, 2H); 6.58 (d, J=9.2 Hz, 2H);3.02 (q, J=7.05 Hz, 8H); 0.93 (t, J=7.04 Hz, 12H)

Ms (FAB): Calculated for C₂₈H₂₉O₃N₂Br₂ (MH)⁺: 599.0545

-   -   Found: 599.0527

Example IX Preparation of 2,7-dibromo Rhodamine B Lactone (20)

To a stirred solution of 2, 7-dibromodihydrorhodamine B methyl ester (3)46 mg (0.10 mmol) in 4 ml of dichloromethane was added chloranil (1.2eq., 0.12 mmol, 30 mg). The reaction mixture was stirred at roomtemperature overnight, then the reaction was stopped and the solvent wasevaporated under reduced pressure to give a purple residue. The oxidizedcompound obtained in the precedent step was dissolved in 4 ml of dioxaneand HCl (1M) (5 ml) was added dropwise, and the resulting solution waswormed in water bath to give a clear red solution. After evaporation todryness under reduced pressure we obtained a purple viscous residue. Theresidue was purified by flash chromatography using a ethyl acetate:hexanes (1.5:8.5) as eluent to give the desired compound 4 as a whitefoam solid (35 mg).

Rf: 0.34 (AcOEt: hexanes 1.5: 8.5)

Yield: 80%/o

Nmr: (CD3OD) δ 7.92 (dd, J=1.45 and 7.5 Hz, 1H) 7.63 (m, 4H); 7.18 (dd,J=1.6 and 7.2 Hz, I H); 7.02 (m, 2H).

Ms (FAB): Calculated for C₂₅H₂₉O₃N₂Br₂ (MH)⁺: 599.0545

Found: 599.0570

Examples of Uses of the Rhodamine Derivatives According to the Inventionas Intermediates Example X X Synthesis of 4-bromo-5-phenyl Rhodamine BMethyl Ester Chloride (22) X-1 Preparation of 4-bromo-5-phenyl RhodamineB Lactone (21)

A stirred mixture of 10 mmole of the dibromolactone 19, 10 mmol ofphenylboronic acid, 4.2 mL (30 mmol) of Et₃N, 0.067 g (0.3 mmole) of Pd(OAc)₂, and either 0.19 g (0.62 mmol) of tri-o-tolylphosphine catalystor 0.16 g (0.62 mmol) of PPh₃ catalyst, in 40 mL of DMF is heated undera nitrogen atmosphere to 100 C for 2-3 hours. The solvent is thendistilled off under reduced pressure, and the residue partitionedbetween CH₂Cl₂ and 10% aqueous NH₃. The organic extracts are then dried(MgSO₄) and concentrated under reduced pressure. Purification by flashchromatography on silica gel affords the pure monobromolactone 21. (SeeW. J. Thompson and J. Gaudino, J. Org. Chem. 1984, 49, 5237-5243; N.Miyaura, T. Yanagi, and A. Suzuki, Synthetic Communications, 1981,11(7), 513-519).

X-2 Preparation of 4-bromo-5-phenyl Rhodamine B Methyl Ester Chloride(22)

Methanolysis and concomitant oxidation of monobromolactone 21 is carriedout by first stirring a mixture of 3-4 mmoles the compound in 100 mL ofmethanol while bubbling in a fine stream of anhydrous HCl gas for aperiod of 45 min and then heating the mixture to reflux overnight. Themethanol is then evaporated under reduced pressure and the dark redresidue purified by flash chromatography to afford the desired dark redproduct 22.

Example XI XI Synthesis of 2,7-dibromo-4,5-dimethyl Rhodamine B MethylEster Bromide (24) XI-1 Preparation of 4,5-dimethyl Rhodamine B Lactone(23)

To a solution of 7.0 mmol of the dibromolactone 19 inhexamethylphosphoramide (HMPA) is added 0.05 mmol of the catalystbenzylbromobis (triphenylphosphine)-palladium (11) and 16.0 mmol oftetramethyltin. The solution is then heated to 65° C. with stirringunder air in a sealed tube until blackening occurs. The solution is thencooled to room temperature and 5 mL of water is added. The mixture isextracted with dichloromethane and the organic solution dried overMgSO₄. Evaporation of the solvent yields the crude product which ispurified by flash chromatography on silica gel to give the pure lactone23. (See D. Milstein and J. K. Stille, J. Amer. Chem. Soc. 1979, 101(17), 4992-4998).

XI-2 Preparation of 2, 7-dibromo-4, 5-dimethyl Rhodamine B Methyl EsterBromide (24)

A solution of 1.25 mmol 4,5-dimethyl Rhodamine B lactone 23 in 50 mlmethanol was treated, at room temperature, by 2.5 mmol of bromine. Aprecipitate was formed after the addition of bromine and the mixture wasstirred for 3 hours. The solvent was evaporated under reduced pressureto give a red solid which was recrystallized from methanol: diethylether to give the desired dibromomethyl ester 24.

Example XII XII Synthesis of 2-bromo-7-ethynyl Rhodamine B Methyl EsterBromide (26) XII-I Preparation of 2-bromo-7-ethymyl Rhodamine B Lactone(25)

A mixture of 53 mmol dibromolactone 20 and 53 mmol ofethynyltrimethylsilane, 300 mg of triphenyl phosphine and 150 mg ofpalladium (II) acetate is prepared in 100 ml of deaerated anhydroustriethylamine at 30-40° C. The mixture is then heated under argon at90-100° C. for 22 hours The mixture is cooled and filtered to give thedesired impure trimethylsilyl derivative of 25. Treatment with potassiumcarbonate at 25° C. for 16 hours followed by neutralization giveslactone 25 after purification by flash chromatography. (See W. B.Austin, N. Bilow, W. J. Kelleghan, and K. S. Y. Lau. J. Org Chem. 1981,46, 2280-2286; S. Takahashi, Y. Kuroyama, K. Sonogashira, N. Hagihara,Systhesis, 1980, 627-630).

XII-2 Preparation of 2-bromo-7-ethynyl Rhodamine B Methyl Ester Chloride(26)

A stirred solution of 3.5 mmol of 2-bromo-7-ethynyl Rhodamine B lactone25 in 100 mL of methanol is treated with a fine stream of bubbled HClgas for 45 min. The reaction mixture is then heated to reflux overnightand the methanol evaporated under reduced pressure. The dark red residueis purified by flash chromatography using a mixture of methanol anddichloromethane to afford the desired ester 26.

Example XIII XIII Synthesis of 4,5-dibromo-2,7-di-n-butyl Rhodamine BMethyl Ester Bromide (28) XIII-1 Preparation of 2,7-di-n-butyl RhodamineB Lactone (27)

A solution of 7.0 mmol of the dibromolactone 20 in 4 mlhexamethylphosphoramide (HMPA) is treated with 0.05 mmol benzylbromobis(triphenylphosphine) palladium (II) and 16.0 mmol of tetra-n-butyltincompound. The solution is then heated to 65° C. with stirring under airin a sealed tube until blackening occurs. The solution in then cooled toroom temperature and 5 ml of water is added. The mixture is extractedwith dichloromethane and the latter is evaporated in vacuo to give thecrude product which is purified by flash chromatography to yield thepure lactone 27. (See D. Milstein and J. K. Stille, J Amer. Chem. Soc.1979, 101(17), 4992-4998).

XIII-2 Preparation of 4,5-dibromo-2,7-di-n-butyl Rhodamine B MethylEster Bromide (28)

A solution of 1.25 mmol of Rhodamine B lactone 27 in 50 ml of methanolis treated, at room temperature, with 2.5 mmol of bromine. A precipitateis formed shortly after the addition of bromine and the mixture isstirred for 3 hours. The solvent is then evaporated under reducedpressure to give a red solid which is recrystallized from methanol:diethyl ether to give the desired dibromomethyl ester bromide 28.

Determination of the Bacteriosidic and/or Bacteriostatic of RhodamineDerivatives

Experimental Design

The following experimental procedures have been used for thedetermination of antibacterial activity.

Bacteriostasis:

Escherichia coli: (0157)a

The protocol used for the bacteriosidic inactivation was performed asdescribed in Brasseur and coll. with few modifications for bacteriosidicpotential assessment (Brasseur et al, 2000). Compounds TH9402, HA-X-44,HA-X-164, HA-X-171 and HA-VIII-92 showed antibacterial activity againstE. coli using the following experimental procedure.

Bacterial was grown overnight in Lubria Broth medium (LB), 100 μl(≈1×10⁷ bacteria) of each bacterial suspension was added to 4 ml of LBmedium, a small aliquot of the bacterial suspension was taken out forbacterial titer prior to treatment expressed as CFU/mL (colony formingunits per mL). To the bacterial suspension was added the rhodaminesderivatives, each derivative being tested in duplicate at aconcentration of 50 μM. Each mixture of bacteria rhodamine derivativewas incubated at 37° C. for 40 minutes. The bacteria-rhodaminesuspensions were then treated and exposed to a 514 nm wavelength lightfor 180 minutes, for a total output energy of 30 Joules/cm². Followingthe treatment time, the bacteria-rhodamine suspensions were centrifugedat 3000 g, resuspended in 4 ml and serial dilutions were performed foreach duplicate. 10 μL of the diluted bacterial suspensions were plated,the plates incubated overnight at 37° C. The bacteriostatic effect isexpressed by the number of CFU/mL.

Pseudomonas aeruginosa:

The protocol used for the bacteriosidic inactivation was performed asdescribed in Brasseur and coll. with few modifications for bacteriosidicpotential assessment (Brasseur et al, 2000). Compound TH9402 showedantibacterial activity against P. aeruginosa using the followingexperimental procedure.

Bacterial was grown overnight in Lubria Broth medium (LB), 100 μl(≈1×10⁷ bacteria) of each bacterial suspension was added to 4 ml of LBmedium, a small aliquot of the bacterial suspension was taken out forbacterial titer prior to treatment expressed as CFU/mL. To the bacterialsuspension was added the rhodamines derivatives, each derivative beingtested in duplicate at a concentration of 50 μM. Each mixture ofbacteria rhodamine derivative was incubated at 37° C. for 40 minutes.The bacteria-rhodamine suspensions were then treated and exposed to a514 nm wavelength light for 180 minutes, for a total output energy of 30Joules/cm². Following the treatment time, the bacteria-rhodaminesuspensions were centrifuged at 3000 g, resuspended in 4 ml and serialdilutions were performed for each duplicate. 10 μL of the dilutedbacterial suspensions were plated, the plates incubated overnight at 37°C. The bacteriostatic effect is expressed by the number of CFU/mL.

Salmonella typhimurium

The protocol used for the bacteriosidic inactivation was performed asdescribed in Brasseur and coll. with few modifications for bacteriosidicpotential assessment (Brasseur et al, 2000). Compounds TH9402, HA-X-44and HA-X-164 showed antibacterial activity against S. typhirrauriufnusing the following experimental procedure.

Bacterial was grown overnight in Lubria Broth medium (LB), 100 μl (≈×10⁷ bacteria) of each bacterial suspension was added to 4 ml of LB medium,a small aliquot of the bacterial suspension was taken out for bacterialtiter prior to treatment expressed as CFU/mL. To the bacterialsuspension was added the rhodamines derivatives, each derivative beingtested in duplicate at a concentration of 50 μM. Each mixture ofbacteria rhodamine derivative was incubated at 37° C. for 40 minutes.The bacteria-rhodamine suspensions were then treated and exposed to a514 nm wavelength light for 180 minutes, for a total output energy of 30Joules/cm². Following the treatment time, the bacteria-rhodaminesuspensions were centrifuged at 3000 g, resuspended in 4 ml and serialdilutions were performed for each duplicate. 10 μL of the dilutedbacterial suspensions were plated, the plates incubated overnight at 37°C. The bacteriostatic effect is expressed by the number of CFU/mL.

Staphilococcus epidermitis

The protocol used for the bacteriosidic inactivation was performed asdescribed in Brasseur and coll. with few modifications for bacteriosidicpotential assessment (Brasseur et al, 2000). Compounds TH9402, HA-X-40,HA-X-44, HA-X-149 and HA-X-164 showed antibacterial activity against S.tryphimurium using the following experimental procedure.

Bacterial was grown overnight in Lubria Broth medium (LB), 100 μl(≈1×10⁷ bacteria) of each bacterial suspension was added to 4 ml of LBmedium, a small aliquot of the bacterial suspension was taken out forbacterial titer prior to treatment expressed as CFU/mL. To the bacterialsuspension was added the rhodamines derivatives, each derivative beingtested in duplicate at a concentration of 50 μM. Each mixture ofbacteria rhodamine derivative was incubated at 37° C. for 40 minutes.The bacteria-rhodamine suspensions were then treated and exposed to a514 nm wavelength light for 180 minutes, for a total output energy of 30Joules/cm². Following the treatment time, the bacteria-rhodaminesuspensions were centrifuged at 3000 g, resuspended in 4 ml and serialdilutions were performed for each duplicate. 10 μL of the dilutedbacterial suspensions were plated, the plates incubated overnight at 37°C. The bacteriostatic effect is expressed by the number of CFU/mL.

Staphilococcus epidermitis

The protocol used for the bacteriosidic inactivation was performed asdescribed in Brasseur and coll. with few modifications for bacteriosidicpotential assessment (Brasseur et al, 2000). Compounds HA-X-171 andHA-VIII-92 showed antibacterial activity against S. epidermitis usingthe same experimental procedure except that a concentration of 10 μM wasused in the experimental procedure.

Bacterial was grown overnight in Lubria Broth medium (LB), 100 μl(≈1×10⁷ bacteria) of each bacterial suspension was added to 4 ml of LBmedium, a small aliquot of the bacterial suspension was taken out forbacterial titer prior to treatment expressed as CFU/mL. To the bacterialsuspension was added the rhodamines derivatives, each derivative beingtested in duplicate at a concentration of 50 μM. Each mixture ofbacteria rhodamine derivative was incubated at 37° C. for 40 minutes.The bacteria-rhodamine suspensions were then treated and exposed to a514 nm wavelength light for 180 minutes, for a total output energy of 30Joules/cm². Following the treatment time, the bacteria-rhodaminesuspensions were centrifuged at 3000 g, resuspended in 4 ml and serialdilutions were performed for each duplicate. 10 μL of the dilutedbacterial suspensions were plated, the plates incubated overnight at 37°C. The bacteriostatic effect is expressed by the number of CFU/mL.

Staphilococcus epidermitis

The protocol used for the bacteriosidic inactivation was performed asdescribed in Brasseur and coll. without modification for bacteriosidicpotential assessment (Brasseur et al, 2000). Compound HA-X-40 showedantibacterial activity against S. epidermitis using the sameexperimental procedure except that an extrusion time of 90 minutes wasperformed prior to radiation treatment.

Bacterial was grown overnight in Lubria Broth medium (LB), 100 μl(≈1×10⁷ bacteria) of each bacterial suspension added to 4 ml of LBmedium. A small aliquot of the bacterial suspension was taken out forbacterial titer prior to treatment. To the bacterial suspension wasadded the rhodamines derivatives, the derivative being tested induplicate at a concentration of 50 μM. Each mixture of bacteriarhodamine derivative was incubated at 37° C. for 40 minutes. Thebacterial suspensions were then centrifuged at 3000 g for 10 minutes,resuspended in 4 mL LB media and incubated for 90 minutes at 37° C. toallow extrusion of the derivatives. The bacteria rhodamines suspensionswere then treated and exposed to a 514 nm wavelength light for 180minutes, for a total output energy of 30 Joules/cm². Following thetreatment time, serial dilutions were performed for each duplicate and10 μL of the diluted bacterial suspension was plated, the platesincubated overnight at 37° C. The bacteriostasic effect is expressed bythe number of colony forming units/mL.

Determination of the Antiviral Activity of the Rhodamine Derivatives ofFormula (I)

Antiviral Assay:

References

-   Lin L., Inactivation of cytomegalovirus in platelet concentrates    using Helix™ technology, Seminar in Hematology, 2001, 38, 4, Supp.    #11, 27-33;-   Brasseur, N., Ménard, I., Forget, A., El Jastimi, R., Hamel, R.,    Molfino, N. A. and E van Lier, J., Eradication of Multiple Myeloma    and Breast Cancer Cells by Th9402-mediatcd Photodynamic Therapy:    Implication of Clinical Ex vivo Purging of Autologous Stem Cell    Transplant, Photochemistry and Photobiology, 2000, 72, 6, 780-878;-   Lin. B., Londe, H., Janda, J. M., Hanson, C. V. and Corash, L.,    Photochemical Inactivation of Pathogenic, Bacteria in Human Platelet    Concentrates, Blood, 1994, 83, 9, 2698-2706;

Lin. B. L., Londe, H., Hanson, C. V., Wiesehahn, G., Isaacs, S., Cimino,G. and Corash, L., Photochemical Inactivation of Cell-Associated HumanImmunodeficiency Virus in Platelets Concentrates, Blood, 1993, 82, 1,292-297;

-   Lin, B. L., Wiesehahn, G. P., Morel, P. A. and Corash L., Use of    8-Methoxypsoralen and Long-Wavelength Ultraviolet Radiation for    Decontamination of Platelet Concentrates, Blood, 1989, 74, 1,    517-525.    Objective:

The antiviral assay was performed as described in Lin, L (2001). Humandiploid fibroblast, foreskin cells (FS), were used in this assay. Theanti-viral activity of rhodamines derivatives were tested and resultsshowed that all compounds, HA-X 40, HA-X-149, HA-X-164, HA-X-171 andHA-VIII-92 followed by PDT treatment possess antiviral activity againstCytomegalovirus.

Method:

FS cells were grown to confluency in shell vials. At the time ofinfection 2.5-3.5×10 cells were growing on each coverslip. The CMV(AD169) stock solution containing 1 mL of virus were quickly thawed,seeded and diluted following 100 fold dilutions in MEM (Earle's salt)supplemented with L-glutamine and 2% FBS, total volume 30 ml.

The titer of the virus have been determined at 10⁻² (10⁴ TCID₅₀) plaqueforming units (pfu) in 0.2 ml. Therefore 1 mL used in the PDTexperiments represents 1.4×10⁵ pfu. A M. O. I. of 0.4-0.5 of CMV wasused throughout this experiment.

The plates containing no rhodamines derivatives were treated with lightin parallel to the non-light treated plates. The concentration usedthroughout the assay for the rhodamines derivatives was maintained at 50uM. Following the addition of the derivatives to the viral stocksolution, the plates were placed into the Theralux L6.30 device andilluminated for 180 minutes with 210 rpm agitation. The energy outputwas measured to be 30 Joulesicm². The non-PDT plate was placed into a37° C. incubator for the same amount of time. Following this treatmenttime, dilutions were made and inoculated with the FS cells undercentrifugation (2000 g, 60 minutes). Following the centrifugation, thecells are incubated 60 minutes at 37° C., 5% CO2, then are washed withthe culture media and incubated for 18-24 hours at 37° C. at 5% CO₂.Inoculation volume of each dilution was 0.2 ml, the dilutions made were10⁻³ to 10⁻⁵ in duplicate.

The cells were fixed, removed from the vials and stained with labelledFITC (fluorescein isothiocyanate) Mab to CMV immediate early antigen.

CMV viral particles were counted. One fluorescent virus particle(kidney-shaped) represents one plaque forming unit.

Here is the protocol for the two other compounds TI-9402 and HA-X-44which inhibit cytometalovirus infectivity without PDT treatment as wellas a new version of the table to be added in the patent.

Objective:

The antiviral assay was performed as described in Lin, L (2001). Humandiploid fibroblast, foreskin cells (FS), were used in this assay. Theanti-viral activity of rhodamines derivatives were tested and resultsshowed that compounds, TH9402 and HA-X-44 did not need PDT treatment topossess antiviral activity against Cytomegalovirus.

Method:

FS cells were grown to confluency in shell vials. At the time ofinfection 2.5-3.5×10 cells were growing on each coverslip. The CMV(AD169) stock solution containing 1 mL of virus were quickly thawed,seeded and diluted following 100 fold dilutions in MEM (Earle's salt)supplemented with L-glutamine and 2% FBS, total volume 30 ml.

The titer of the virus have been determined at 10⁻² (10⁴ TCID₅₀) plaqueforming units (pfu) in 0.2 ml. Therefore 1 mL used in the PDTexperiments represents 1.4×10⁵ pfu. A M. O. I. of 0.4-0.5 of CMV wasused throughout this experiment.

The plates containing no rhodamines derivatives were treated with lightin parallel to the non-light treated plates. The concentration usedthroughout the assay for the rhodamines derivatives was maintained at 50uM. Following the addition of the derivatives to the viral stocksolution, dilutions were made and inoculated with the FS cells undercentrifugation (2000 g, 60 minutes). Following the centrifugation, thecells are incubated 60 minutes at 37° C., 5% CO2, then are washed withthe culture media and incubated for 18-24 hours at 37° C. at 5% CO₂.Inoculation volume of each dilution was 0.2 ml, the dilutions made were10⁻³ to 10⁻⁵ in duplicate.

The cells were fixed, removed from the vials and stained with labelledFITC (fluorescein isothiocyanate) Mab to CMV immediate early antigen.

CMV viral particles were counted. One fluorescent virus particle(kidney-shaped) represents one plaque forming unit.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features herein before set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. A method for treating a tissue sample from a patient, comprising the steps of: harvesting the tissue sample from the patient, wherein the tissue sample is infected with bacteria, wherein the bacteria is gram positive; providing a rhodamine compound, wherein the rhodamine compound is 2′-(6-dimethylamino-3-dimethylimino-3H-xanthen-9-yl) 4′, 5′-dichloro-benzoic acid methyl ester hydrochloride; mixing the rhodamine compound and the tissue sample to form a mixture, exposing the mixture to radiant energy to inhibit or kill the bacteria; and transplanting the exposed mixture into the patient.
 2. The method of claim 1, wherein the bacteria is Staphylococcus epidermitis.
 3. The method of claim 1, wherein the mixture is exposed to a wavelength of 514 nm.
 4. The method of claim 1, wherein the tissue sample is blood.
 5. The method of claim 1, wherein the tissue sample is bone marrow.
 6. The method of claim 1, wherein the mixture is exposed to radiant energy for 180 minutes.
 7. The method of claim 1, further comprising the steps of centrifuging the mixture; resuspending the centrifuged mixture; and incubating the resuspended centrifuged mixture.
 8. The method of claim 7, wherein the resuspended centrifuged mixture is incubated for 90 minutes.
 9. The method of claim 7, wherein the resuspended centrifuged mixture is incubated before exposing the mixture to radiant energy. 